tRNA derived small RNAs (tsRNAs) involved in cell viability

ABSTRACT

The present invention features compositions and methods relating to tRNA-derived small RNAs (tsRNAs). Provided herein are oligonucleotide compositions that are complementary to tsRNAs, in particular leuCAGtsRNA, and methods of using the oligonucleotides for the regulation of respective tsRNA. Further provided are methods of inducing apoptosis through the inhibition of leuCAGtsRNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/216,546, filed Jul. 21, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/214,032, filed Mar. 14, 2014, now U.S. Pat. No.9,428,537, which claims the benefit of U.S. Provisional Application No.61/798,871, filed Mar. 15, 2013, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under contract DK78424awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

tRNA-derived small RNA (tsRNA) are small RNAs that are derived from thecleavage of tRNA. tRNA fragments of 30-35 nucleotides have beenidentified in bacteria, fungi, plants, and animals (Lee and Collins, J.Biol. Chem., 280:42744-9 (2005); Haiser et al. Nucleic Acids Res.36:732-41 (2008); Jochl et al. Nucleic Acids Res. 36:2677-89 (2008);Kawaji et al BMC Genomics 9:157 (2008); Li et al. Nucleic Acids Res.36:6048-55 (2008); Thompson et al. RNA 14:2095-103 (2008); and Zhang etal. Plant Physiol. 150:378-87 (2009)). There are numerous differenttsRNAs in the cell and the functions of how each of these small RNAsinteract and function with other cellular components is not completelyknown.

Mature tRNAs are essential for mRNA translation in their role oftransferring amino acids to a growing polypeptide chain. However, tRNAfragments have recently been identified as a source of non-coding RNAs(reviewed in (Martens-Uzunova et al., 2013; Sobala and Hutvágner,2011)). tRNA fragments are classified into two classes based on theirsizes. The longer 30-35 nt RNA species are called tRNA halves andgenerated by the endonuclease angiogenin. There is growing evidence thattRNA halves are involved in cellular stress response, cellproliferation, and apoptosis.

The other 18-26 nt non-coding RNAs are called small tRNA fragments (tRF)or tRNA-derived small RNA (tsRNA), which have been classified into threegroups: 5′tsRNA (tRF-5 or 5′tRF), type I tsRNA (3′tsRNA, tRF-3, or3′CCAtRF), and type II tsRNA (tRF-1 or 3′U tRF) (Haussecker et al.,2010; Lee et al., 2009). The 5′ and 3′tsRNAs are derived from the 5′ and3′ end of mature tRNAs, respectively. The 3′tsRNA contains the CCAsequence added to 3′end during tRNA maturation. The tsRNA type II isprocessed from the 3′ precursor of tRNA, which ends in polyuridine dueto termination by RNA polymerase III.

The biogenesis of tsRNAs is not clear. For the generation of type IItsRNAs and 5′tsRNAs there are mixed reports supporting the role ofdicer, and one study supporting the role of the tRNA processing enzymeRNaseZ and tRNA 3′-endonuclease, ELAC2, in type II generation (Babiarzet al., 2008; Cole et al., 2009; Haussecker et al., 2010; Lee et al.,2009), while the generation of the 3′tsRNAs (type I) is unlikely relatedto dicer processing (Babiarz et al., 2008; Li et al., 2012).

The biological role of tsRNAs is not well understood and there have beenattempts to establish whether tsRNAs are associated with Ago (Argonaute)proteins, the key component in RISC (RNA-induced silencing complex)(reviewed in (Bartel, 2004; Croce and Calin, 2005; Kim and Kim, 2012;Pederson, 2010)). The evidence for the presence of tsRNA in RISC comesfrom studies showing that certain tsRNAs can associate withover-expressed Argonaute proteins (Haussecker et al., 2010; Maute etal., 2013). Furthermore, HisGTG and LeuCAG3′tsRNA as well asGlyGCC3′tsRNA have been found to be associated with endogenous Ago2protein (Li et al., 2012; Maute et al., 2013). In addition, there issome implication that the over-expressed GlyGCC3′tsRNA from a miRNAhairpin or genomic tRNA can reduce endogenous gene expression throughbase-pairing with complementary target mRNAs in the 3′UTR (Maute et al.,2013). Synthetic S′tsRNAs can inhibit protein translation regardless oftheir ability to base-pair with complementary target mRNAs, implyingthat the cellular function of S′tsRNA differs from microRNA (Sobala andHutvágner, 2013).

tRNA-derived small RNAs are also found in lower organisms. InTetrahymena, a 18-22 nt fragment of the 3′tRNA is associated with Twi12(Tetrahymena Piwi12), which is essential for cell growth, and does nothave trans-gene silencing activity (Couvillion et al., 2010). Twi12activates Xrn2 for RNA processing in the nucleus (Couvillion et al.,2012). In Haloferax volcanii, the Val5′tsRNA binds to the ribosome, anda synthetic Val5′tsRNA was shown to inhibit translation (Gebetsberger etal., 2012). All of these various findings suggest that some of thesetsRNAs play important roles in various aspects of cellular function.

Unlike the type II tsRNAs, the 5′tsRNAs and 3′tsRNAs (type I tsRNAs) arederived from mature tRNAs making their sequences more highly conservedbetween species. As reported herein, these tsRNAs play an important rolein cell viability. Specifically, when tsRNAs are depleted, cells undergoapoptosis.

Apoptosis is a genetically programmed cellular event that ischaracterized by well-defined morphological features, such as cellshrinkage, chromatin condensation, nuclear fragmentation, and membraneblebbing. Kerr et al. Br. J. Cancer, 26, 239-257 (1972); Wyllie et al.Int. Rev. Cytol., 68, 251-306 (1980). It plays an important role innormal tissue development and homeostasis, and defects in the apoptoticprogram are thought to contribute to a wide range of human disordersranging from neurodegenerative and autoimmunity disorders to neoplasms.Thompson, Science, 267, 1456-1462 91995); Mullauer et al. Mutat. Res,488, 211-231 (2001).

For example, as reported herein, cells in which LeuCAG3′tsRNA isdepleted undergo apoptosis by an unusual mechanism that involvestsRNA-mediated depletion of 40S ribosomal subunits. Thus, methods ofregulating tsRNAs can be used to regulate apoptosis and control disease.

BRIEF SUMMARY OF THE INVENTION

In various embodiments, the present invention provides anoligonucleotide that is complementary to a tRNA-derived small RNA(tsRNA) comprising at least one locked nucleic acid. In an embodiment,the oligonucleotide is of a structure according to Formula (I).(A)_(x)-(B)_(y)-(C)_(z)  Formula (I)wherein x, y, and z are integers that are greater than or equal to 1, Ais a locked or unmodified nucleic acid. When x is greater than 1, each Ais independently selected and A is the 5′ end of the oligonucleotide.When B is a locked or unmodified nucleic acid and y is greater than 1,each B is independently selected. When C is a locked or unmodifiednucleic acid and z is greater than 1, each C is independently selectedand C is the 3′ end of the oligonucleotide. In an exemplary embodiment,A and C are locked nucleic acids, and B is one or more unmodifiednucleic acids.

In an exemplary embodiment, the oligonucleotide is selected from thegroup consisting of: (a) tGTcAGgAgTggGaT (SEQ ID NO: 2); and (b)GGTGtcaggagtggGATT (SEQ ID NO: 11), where the uppercase lettersrepresent locked nucleic acids and lowercase letters representunmodified nucleic acids. In an exemplary embodiment, theoligonucleotide is complementary to a tsRNA molecule or a 3′ end of amature tRNA molecule. In an exemplary embodiment, the oligonucleotide iscomplementary to a group selected from leucine-CAG tsRNA and leucine-CAGtRNA. In an exemplary embodiment, the oligonucleotide comprises apharmaceutically acceptable carrier.

In various embodiments, the present invention provides a method ofinhibiting viability of a cell, the method comprising administering tothe cell an oligonucleotide of the present invention. An exemplarymethod comprises inhibiting the function of leucine-CAG tsRNA. In anexemplary embodiment, the method inhibits cell proliferation, inducesapoptosis, or induces cellular necrosis. In an exemplary embodiment, theinhibiting induces apoptosis.

In various embodiments, the present invention provides a methodcomprising treating a disease in a subject, the method comprisingadministering to the subject an oligonucleotide of the presentinvention. In an embodiment, the disease is one or more of cancer,autoimmune disease, a non-malignant state, or an excessive vascularstate. In an embodiment, the non-malignant state is a hyperplasia andthe excessive vascular state is macular degeneration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an overview of tRNA-derived small RNAs.

FIGS. 2A illustrates target sequences of oligonucleotides comprisinglocked nucleic acids (LNA) against leuCAG mature tRNA. Actualoligonucleotides are complementary to target sequences. Bold andunderlined characters are LNA and remaining characters represent DNA.Boxed characters are LNA mis-match of leu3tsRNA. FIG. 2B shows thetarget sequences of LNA-containing oligonucleotides. Actualoligonucleotides are complementary to target sequences. Table 6 providesthe representative sequence identifiers. FIG. 2C shows inhibition ofleuCAG3tsRNA by the leu3ts GAPmer oligonucleotide (SEQ ID NO: 11).Mature leu tRNA is used as loading control.

FIGS. 3A-3H show that the depletion of LeuCAG3′tsRNA impairs cellviability and illustrates inactivation of tsRNA by mixmeroligonucleotides. FIG. 3A shows the sequence and predicted structure ofmature human LeuCAG tRNA. The underlined sequences are the target ofeach indicated LNA. Closed and open triangles indicate 5′ end ofLeuCAG3′tsRNA and 3′end of LeuCAG5′tsRNA, respectively. FIG. 3B showsthe depletion of 3′tsRNA (CAGPM) from LeuCAG impairs HeLa and HCT-116cell viability, whereas depletion of 3′tsRNA from AspGTC (AspPM), SerGCT(SerPM), MetCAT tRNA (MetiPM) does not. To determine cell viability, 3days post-transfection, colorimetric cell proliferation assay (MTSassay) was performed. X-axis is transfected LNA and y-axis is therelative MTS value normalized to the MTS value from GL2 transfection asa control. FIG. 3C shows that LNA directed against the 3′tsRNA does notaffect mature tRNAs. LNA directed against the anticodon loop (CAGcodon)or 5′end (CAGS'tsPM) of LeuCAG tRNA as well as LeuCAG3′tsRNA withdifferent 2nt mismatches (CAGMM or CAGMM2) does not affect HeLa andHCT-116 cell viability. Cell viability was determined as in FIG. 3B.FIG. 3D shows the specific down-regulation of tsRNA by mixmer LNAoligonucleotides. Specifically, FIG. 3D illustrates the down regulationof leuCAG3tsRNA by leu3ts15PM (CAGPM) (SEQ ID NO: 2). LNA knockdown ofLeuCAG3′tsRNA occurs in a sequence specific manner. After transfectionof indicated LNA for 24 hrs, total RNA was subjected to northernblotting. LeuCAG3′tsRNA is bound to CAGPM and is not detected by theprobe. U6 snRNA is the loading control. FIG. 3E shows gapmer LNAs, whichinduce RNase H activity, sequence specifically cleaves LeuCAG3′tsRNA,whereas the mature tRNA is not cleaved. After transfection of indicatedgapmer LNAs for 24 hrs, northern hybridization was done as in FIG. 3D.FIG. 3F shows that depletion of LeuCAG3′tsRNA decreased the number ofviable HeLa cells. Day post-The cell number normalized to day zeroversus day post transfection. The normalized value at day 0 was set at100. FIG. 3G shows that single stranded LeuCAG3′ts 18bp and 21bp rescuesthe LNA-induced decrease in cell viability. LNAs (GL2 (black bar) orCAGPM (grey bar)) and indicated single-stranded tsRNA were separatelytransfected into HeLa cells for 72 hrs. X-axis is single-stranded tsRNA;y-axis is the relative MTS value normalized to the MTS value from eachGL2 transfection as a control. Cell viability was determined as in FIG.3B. Each experiment was performed in triplicate and repeated twice.*P<0.01 compared with GL2 transfected cells untreated; GL2, control LNAcomplementary to firefly luciferase gene from pGL2 vector (Elbashir etal., 2001); CAGPM, LNA complementary to LeuCAG3′tsRNA; CAGMM, LNAcomplementary to LeuCAG3′tsRNA with 2 nt mis-match; CAGMM2, LNAcomplementary to LeuCAG3′tsRNA with 2 nt mis-match (mis-match positionis different from CAGMM); CAG5′tsPM, LNA complementary to LeuCAG5′tsRNA;CAGcodon, LNA complementary to LeuCAG tRNA anti-codon; AspPM, LNAcomplementary to 3′ end of AspGTC tRNA; SerPM, LNA complementary toSerGCT tRNA; MetiPM, LNA complementary to MetCAT initiator tRNA;Gap_GL2, gapmer LNA complementary to firefly luciferase gene;Gap_5′tsPM, gapmer LNA complementary to LeuCAG5′tsRNA; Gap_codonPM,gapmer LNA complementary to LeuCAG tRNA codon; Gap_3′tsPM, gapmer LNAcomplementary to LeuCAG3′tsRNA. Error bars represent the standarddeviation. FIG. 3H illustrates the specific down-regulation of tsRNA bymixmer LNA oligonucleotides, specifically the down regulation ofserGCT3tsRNA by Ser15GCTPM oligonucleotide (SEQ ID NO: 7).

FIG. 4 illustrates that leu3ts15PM (CAGPM) does not function throughcanonical miRNA/RNAi pathway. The graph shows normalized Renillaluciferase activity. SCRinCodonN represents scramble sequences that arelocated 6 bp downstream from the ATG start codon of the Renillaluciferase gene. CAGinCodonN represents perfect complementary sequenceslocated 6bp downstream from the ATG start codon of the Renillaluciferase gene. SCRin3UTR represents scramble sequences that arelocated in 3′UTR of Renilla luciferase gene. CAGin3UTR representsperfect complementary sequences of SEQ ID NO: 2 located in the 3′UTR ofthe Renilla luciferase gene. LNACAG15PM is SEQ ID NO:2.

FIG. 5 illustrates that leuCAG3tsRNA does not have trans-gene silencingactivity similar to microRNA. All constructs have 2 copies of perfectcomplementary sites of 18 bp of leuCAG3tsRNA. codon(II): leuCAG 3tsRNAbinding sites in N-terminal codon; codonC: leuCAG 3tsRNA perfectcomplementary sites in C-terminal codon; scr_3utr: scramble sequence in3utr; CAG_3utr: leuCAG 3tsRNA perfect complementary sites in 3′UTR;CAG_5utr: leuCAG 5tsRNA binding sites in 5′UTR

FIG. 6 illustrates that an LNA15bp mixmer does not affect endogenousgenes with similar LNA target sequences.

FIG. 7 illustrates that inactivation of leuCAG3tsRNA by LNA mixmerimpairs cell viability. A) Inactivation of leuGAC3tsRNA impairs HeLacell viability. X axis is transfected LNA and y axis is normalized MTTvalue. B) Inactivation of leuCAG3tsRNA impairs HCT cell viability. Xaxis is transfected LNA and y axis is normalized MTT value. C and D)Inactivation of leuCAG3tsRNA impairs cell viability. X axis istransfected LNA and y axis is normalized MTT value.

FIGS. 8A and 8B illustrate the inactivation of leuCAG3tsRNA and theresulting apoptosis. FIG. 8A illustrates that inactivation ofleuCAG3tsRNA by LNA mixmer impairs cell viability. X axis is the day atwhich the cell number was counted; Y axis is the relative cell numbernormalized by cell number at day 0. FIG. 8B illustrates the induction ofapoptosis from leuCAG3tsRNA inactivation as shown by PARP cleavage.CAG15PM is SEQ ID NO: 2.

FIGS. 9A-9D show that the depletion of LeuCAG3′tsRNA induces apoptosis.FIGS. 9A and 9B show the depletion of LeuCAG3′tsRNA increased cellpopulations undergoing apoptosis. Apoptosis in HeLa cells was measuredusing Annexin V-FITC and PI (propidium iodide) staining every 24 hpost-transfection. FIG. 9A shows a representative result of theapoptosis assay wherein the number in each gate represents thepercentage of cells in the gate. Accordingly, FIG. 9A illustrates thatinactivation of leuCAG3tsRNA induces apoptosis. FIG. 9B shows arepresentative graph of each cell population from FIG. 9A. Values aremeans +/−SD (n=3). GL is GL2c, MM is CAGMM1, PM is CAGPM from Table 6.FIG. 9B shows an average cell population of apoptosis assay done intriplicate. Healthy cells are stained with neither Annexin V nor PI(Q1), early apoptotic cells are stained with only Annexin V (Q4), lateapoptotic cells are stained with both Annexin V and PI (Q3), and deadcells are stained with only PI (Q2). Annexin V and PI staining indicatesthat the HeLa cell population undergoing early apoptosis (Q4) and lateapoptosis (Q3), increased in days following transfection of CAGPM. FIG.9C shows that depletion of LeuCAG3′tsRNA causes DNA fragmentation,another apoptosis marker. TUNEL assay in HeLa cells was performed at 24h post LNA transfection. DNase I is a positive control. Nuclei werestained with DAPI (blue). TUNEL positive cells are shown in red. Mergeis a merged image of DAPI and TUNEL staining. FIG. 9D shows thatdepletion of LeuCAG3′tsRNA causes PARP protein. Total protein extractsfrom HeLa and HCT-116 cells were prepared 24 h post-transfection,separated on 4-12% SDS PAGE and analyzed with indicated antibodies. Theclosed triangle indicates 116 kDa PARP protein, the open triangleindicates 89 kDa cleaved PARP protein. GAPDH is the loading control.

FIGS. 10A and 10B illustrate TUNEL assays 24 and 48 hours posttransfection. FIGS. 10A and 10B present three columns (column from leftto right: DIC, DAPI and TUNEL). The term “GL2c” refers to control LNAtargeting a sequence not represented in HCT116 cells. The term“leu3ts-MM” (CAGMM1) refers to LNA bearing two mismatches withLNA3ts-PM. The term “LNA3ts-PM” (CAGPM) refers to LNA with complementarysequence of leutsRNA. The term “DNase I” refers to the positive control.

FIG. 11 illustrates cell cycle analysis of BrdU against propoidiumiodide staining. The bottom portion illustrates cell cycle analysisusing flow cytometry showing that the G1 phase is accumulated in HCTcells after inactivation of leuCAG3tsRNA. The top portion illustrates arepresentative graph of each cell population. PM is CAGPM as listed inTable 6.

FIGS. 12A and 12B illustrate that global protein synthesis is notrepressed by inactivation of leuCAG3tsRNA in HeLa or HCT116 cells 16hours post transfection. Untreated represents untreated cells and mockrepresents cells treated with lipofectamine alone. 15 MM is CAGMM1; 15PMis CAGPM as listed in Table 6. FIG. 12A shows images of HeLa cells. FIG.12B shows images of HCT116 cells.

FIGS. 13A-13C show that depletion of LeuCAG3′tsRNA does not affectglobal protein synthesis and the function of mature LeuCAG tRNA. FIG.13A shows a global protein synthesis assay using Click-iT® AHA AlexaFluor® 488 assay in HeLa cells was performed 24 h post-transfection ofindicated LNA. The nucleus was stained with DAPI, blue color. Proteinsynthesis was measured with Click-iT® AHA, green color. Merge representsthe merged image with DAPI and Click-iT® AHA. Un, untreated cells; mock,transfection of Lipofectamine 2000 without LNA; CHX,cycloheximide-treated positive control. Each used LNA is described inFIG. 3. FIG. 13A shows that global protein synthesis is not repressed byinactivation of leuCAG3tsRNA (PM is CAGPM as listed in Table 6). FIG.13B shows a lobal protein synthesis assay using [35S]-methioninemetabolic labeling in HeLa cells. 24 h post-transfection. HeLa cellswere grown in media including [35S]-methionine for 10 minutes. Cellswere lysed and equal amount of proteins were resolved on 4-12% SDS PAGE,stained with Coomassie brilliant blue (left) as a loading control; gelswere scanned to measure incorporated radioactivity (right) showing thenewly synthesized protein. Each cell number multiplied by 10⁵ representsthe number of cell plated on 6 well culture dish at 24 hrs prior totransfection. Error bars represent the standard deviation. FIG. 13Cshows that depletion of LeuCAG3′tsRNA does not affect the ability ofmature LeuCAG tRNA to decode the CUG codon (mRNA) to Leucine (aminoacid). Luciferase assay was done 24 h after co-transfection of indicatedLNA and luciferase plasmid. CUG plasmid has unmodified Renilla andfirefly luciferase gene. The CUC plasmid contains the unmodified Renillaand codon-modified firefly gene where all eleven CUG codons werereplaced with CUC codons. Renilla luciferase activity was normalized tofirefly luciferase activity expressed from the same plasmid, and wasnormalized to Renilla/firefly activity from the GL2 controltransfections. Each value is a mean of three transfections. X-axis istransfected LNA; y-axis is normalized Renilla luciferase activity. Errorbars represent the standard deviation.

FIGS. 14A and 14B illustrate codon modification of luciferase assay inHeLa and HCT116 cells. FIG. 14A shows data from HeLa cells. FIG. 14Bshows data from HCT116 cells. Mock represents cells treated withlipofectamine alone. Original (left bar of each set) represents originalpsiCHECK2 dual-luciferase construct. Modified (right bar of each set)represents psiCHECK2 construct with leucine codon CAG switched to AAG orGAG.

FIGS. 15A-15C show that LeuCAG3′tsRNA does not have trans-gene silencingactivity. FIG. 15A shows that LeuCAG3′tsRNA does not repress luciferasegene expression containing two copies of perfect complementary targetsites in its 3′UTR or 5′UTR of firefly gene from pGL3 plasmid. Aluciferase assay was performed 24h after co-transfection of either LNAGL2 or CAGPM, indicated firefly luciferase plasmid (pGL3), and Renillaluciferase plasmid (pRL). Each value from firefly luciferase plasmid wasfirst normalized to the Renilla luciferase value from the co-transfectedRenilla luciferase plasmid, and was normalized to the normalizedfirefly/Renilla value from a scrambled control plasmid co-transfectedwith either GL2 or CAGPM LNA. Each value is a mean of threetransfections. X-axis is the target sequence; y-axis is normalizedfirefly luciferase activity. Scramble, scrambled sequences in 3′UTR, anegative control; LeuCAG3′tsPM in 3′UTR, two copies of perfectcomplementary sequences of LeuCAG3′tsRNA in 3′UTR; LeuCAG3′tsPM in5′UTR, two copies of perfect complementary sequences of LeuCAG3′tsRNA in5′UTR; Let-7 PM is a positive control of luciferase assay, a perfectcomplementary sequences of Let-7 in 3′UTR of firefly gene whoseluciferase activity is repressed by endogenous Let-7 in HeLa cells.Error bars represent the standard deviation. FIG. 15B shows thatLeuCAG3′tsRNA does not affect global gene expression. Scatter plotscomparing gene expression (loge (FPKM+1)) of two RNA-Seq datasets fromsamples 24 h after transfection of indicated LNA. Pearson correlationcoefficient is indicated by an r on each plot. FIG. 15C shows a subsetof 5′tsRNA and 3′tsRNA does not co-sediment with polysomal fractions andmRNA undergoing active translation. Cytoplasmic lysates from HeLa cellswere treated with cycloheximide and separated by ultracentrifugation in10-50% sucrose gradients. Total RNAs were extracted from each fraction,separated on denaturing 15% acrylamide gel, transferred to membrane, andnorthern hybridization was performed. Each northern was performed as aduplicate. Left upper graph is ribosomal profile detected at 254 nm UVshowing sucrose gradient discriminate 40S subunit, 60S subunit, 80Sribosome, and polysomes across sucrose gradient. The open triangle ineach northern blot indicates detected tsRNA.

FIGS. 16A-16E show that LeuCAG3′tsRNA is required for ribosomebiogenesis. FIGS. 16A and 16B show that depletion of LeuCAG3′tsRNAdecreases amount of 40S ribosomal subunits and 80S ribosomes. FIG. 16Ashows that the polysome profile was analyzed 24 h post-transfection ofindicated LNA. Cytoplasmic lysates from HeLa cells were treated withcycloheximide and separated in 10-50% sucrose gradients. FIG. 16B showsthat HeLa cells were treated with 2 mM puromycin for 15 min on ice and10 min at 37° C., and processed as in FIG. 16A. FIG. 16C shows thatdepletion of LeuCAG3tsRNA decreases steady-state levels of 18S rRNA.Northern hybridization was performed with total RNA from HeLa cellsprepared at 24 h post-transfection. Each number on top of imagerepresents the cell number plated on 6-well culture dish the day priorto transfection. FIG. 16D shows that pre-rRNA processing pathways inhuman cells. 45S primary transcript (pre-45S) is divided into 5′external transcribed spacers (5′ETS), mature 18S rRNA, internaltranscribed spacer 1 (ITS1), mature 5.8S rRNA, internal transcribedspacer 2 (ITS2), mature 28S rRNA, and 3′ external transcribed spacers(3′ETS). There are two alternative processing pathways. Depletion ofLeuCAG3′tsRNA prohibits processing from the 30S intermediate to 21Sintermediate form in pathway B. Arrowhead and number indicate cleavagesites. Adapted from (Choesmel et al., 2008; Hadjiolova et al., 1993).FIG. 16E shows that depletion of LeuCAG3′tsRNA suppressed removal of the5′-external transcribed spacer (5′-ETS) during 18S biogenesis. Northernhybridization was performed with total RNA from HeLa cells prepared at24 h after transfection of indicated LNA and siRNA. ITS1 probe detects45S primary transcript and intermediate form of mature 18S rRNAincluding the 41S, 30S, 21S, and 18S-E intermediate forms. 5′ETS probedetects 45S primary transcript and 30S intermediate form of mature 18SrRNA. ITS2 probe detects 45S, and 12S and 28S intermediate form ofmature 5.8S and 28S rRNA.

FIGS. 17A-17E show that depletion of LeuCAG3′tsRNA down-regulates RPS28protein level without affecting its mRNA level, whose depletion causesapoptosis. FIG. 17A shows that depletion of LeuCAG3′tsRNA down-regulatesRPS23 and RPS28 protein level. HeLa cells were transfected withindicated LNA for 24 h and protein levels were determined byimmunoblotting. GAPDH is the loading control. The relative signal toeach GL2 transfection is listed. FIG. 17B shows that depletion ofLeuCAG3′tsRNA does not change nuclear-cytoplasmic subcellularlocalization of RPS6 and RPS28 proteins. HeLa cells were transfectedwith indicated LNA for 24h and each protein level was determined byimmunoblotting. Histone3 and c-Myc proteins are localized to thenucleus. GAPDH and tubulin are found in the cytoplasm. Total, totalextracts; C, cytoplasm; N, nucleus. FIG. 17C shows that decreased RPS28protein level induces apoptosis. HeLa cells were transfected withindicated siRNA for 24 h and 89 kDa, cleaved PARP protein was determinedby immunoblotting. FIG. 17D shows that depletion of LeuCAG3′tsRNA doesnot alter RPS28 mRNA levels. Real time PCR was performed with total RNApurified at 24 h after transfection of indicated LNA. Each mRNA levelwas normalized by GAPDH mRNA. FIG. 17E shows that RPS28 over-expressionrestores 18S ribosomal RNA processing. After co-transfection ofindicated plasmid (GFP expression plasmid or RPS28 expression plasmid)and LNA with HCT-116 cell lines for 24 h, total RNA and protein wereextracted. Northern hybridization was performed with ITS1 probe and 18SrRNA probe (left). Western blotting was performed (right). GFPexpression plasmid is the control.

FIGS. 18A-18D show that the depletion of LeuCAG3′tsRNA suppresses RPS28translation in elongation phase. FIG. 18A shows that depletion ofLeuCAG3′tsRNA specifically alters distribution of RPS28 mRNA within thesucrose gradient. The polysome profile was analyzed 24 hpost-transfection. Cytoplasmic lysates from HeLa cells were treated withcycloheximide and separated in 10-50% sucrose gradients. Total RNAs wereextracted from each fraction, separated on denaturing 0.9% agarose gel,transferred and northern hybridization was performed. The indicated basepairs (bp) provided to the left of the labeled gene name indicates thesize of the coding sequences. The polysome profile is the same as shownin 16A. Methylene blue staining picture (Top) indicates the fractionscontaining 40S, 60S, 80S, and polysome. FIG. 18B shows that relativedistribution of mRNA populations of FIG. 18A across sucrose gradient.Amounts of mRNAs for each fraction were normalized using the sum of themRNA across all fractions. X-axis is the fraction number; y-axis is % ofmRNA abundance. FIG. 18C shows that Harringtonine treatment afterdepletion of LeuCAG3′tsRNA stalls RPS28 mRNA at the 80S monosomesuggesting that RPS28 translation is suppressed during elongation phase.The polysome profile was analyzed 24 h after transfection of LNAfollowed by treatment of harringtonine. Lysate preparation, separationin sucrose gradient, and northern were processed as in FIG. 18A. FIG.18D shows a model for apoptosis resulting from LeuCAG3′tsRNA. Theremoval of the tsRNA results in a slowing of RPS28 mRNA elongation.Partial loss of RPS28 protein inhibits ribosomal RNA maturation. As aresult, fewer 40S ribosomal subunits are produced, inducing cellularapoptosis.

FIGS. 19A-19E show that depletion of LeuCAG3′tsRNA impairs cellviability. FIGS. 19A and 19B shows images that are representative scansof the plates after the MTT assay (FIGS. 3B and 3C) at 3 daypost-transfection respectively. FIG. 19C shows that each LNA directed to3′tsRNA portion of mature tRNA sequence specifically binds to theirintended target. LeuCAG tRNA (Leu) is detected by a probe that wasdesigned to detect the 3′tsRNA (3′tsRNA probe). The same is true for theSerGCT (Ser) and MetCAT (Meti). 5′tsRNA probes used to detect the5′tsRNA (5′tsRNA probe). After transfection of the indicated LNA for 24hrs, total RNA was resolved on 15% acrylamide gel, transferred ontomembrane, and hybridized with the indicated ³²P labeled oligonucleotideprobe. U6 is a loading control. Based on the result in FIG. 19D, the LNAactually binds to denatured mature tRNA during RNA extraction, and notthe highly structured tRNA inside cells. FIG. 19D shows that the LNAmixmer binds to mature tRNA during the extraction of total RNA. GL2 andCAGPM LNA were not transfected, instead, both of them (RNA+GL2 orRNA+CAGPM) were mixed during the extraction of total RNA from HeLacells. Each RNA was detected as in FIG. 19C. FIG. 19E shows thatLeuCAG3′tsRNA knockdown decreased viable HCT-116 cells number. Thex-axis is the day at which the cell number was countedpost-transfection; y-axis is the relative cell number normalized by cellnumber at day 0.

FIG. 20 shows that depletion of LeuCAG3′tsRNA does not affect globalprotein synthesis and the function of mature LeuCAG tRNA. Global proteinsynthesis assay using Click-iT® AHA Alexa Fluor® 488 assay in HCT-116cells was performed at 24 hrs after transfection of indicated LNA. Thenucleus was stained with DAPI, blue color. Protein synthesis wasmeasured with Click-iT® AHA, green color. Merge is merged image withDAPI and Click-iT® AHA. Un, untreated cells; mock, transfection ofLipofectamine 2000 without LNA; CHX, cycloheximide treated positivecontrol. Each LNA is described in the legend to FIG. 3.

FIGS. 21A and 21B show that depletion of LeuCAG3′tsRNA inducesapoptosis. FIG. 21A shows that the knockdown of LeuCAG3′tsRNA increasedcell population undergoing apoptosis. Apoptosis in HCT-116 cells wasmeasured using Annexin V-FITC and P1 (propidium iodide) staining at 24 hpost-transfection. Average cell population of triplicate apoptosisassays. FIG. 21B shows that the knockdown of LeuCAG3′tsRNA causes DNAfragmentation, one of the hallmark of apoptosis. A TUNEL assay inHCT-116 cells was performed at 24 h (left) or 48 h (right)post-transfection. DNase I is a positive control. The nucleus wasstained with DAPI, blue color. Tunel positive cells are stained as redcolor. Merge is a merged image of DAPI and TUNEL staining.

FIGS. 22A and 22B show that LeuCAG3′tsRNA does not have trans-genesilencing activity. FIG. 22A shows that AspGTC3′tsRNA and SerGCT3′tsRNAdo not repress luciferase gene expression containing two copies ofperfect complementary target sites in its 3′UTR, respectively. Aluciferase assay was performed as in FIG. 15A. X-axis, target sites in3′UTR; y-axis, normalized luciferase activity as in FIG. 15A. FIG. 22Bshows that LeuCAG3′tsRNA does not regulate PRDM10 gene expressionthrough binding to 5′UTR of PRDM10 and 3′UTR of PRDM10. A luciferaseassay was performed as in FIG. 15A. X-axis, target sites in 3′UTR;y-axis, normalized luciferase activity as in FIG. 15A.

FIGS. 23A and 23B show that LeuCAG3′tsRNA is required for ribosomebiogenesis. FIG. 23A shows that the knockdown of LeuCAG5′tsRNA (5′end ofLeuCAG tRNA), SerGCT3′tsRNA (3′end of SerGCT tRNA), and MetiCAT3′tsRNA(3′end of MetiCAT tRNA) does not change the ribosome/polysomal profiles.24 h post-transfection cytoplasmic lysates from HeLa cells were treatedwith cycloheximide and separated in 10-50% sucrose gradients. Thepolysomal profile was analyzed as in FIG. 15C. FIG. 23B shows that theknockdown of LeuCAG3′tsRNA suppressed 5′-external transcribed spacer(5′-ETS) processing in 18S biogenesis. Northern hybridization wasperformed with total RNA from HCT-116 and 293T cells prepared at 24 hpost-transfection, ITS1 probe detects 45S primary transcript andintermediate form of mature 18S rRNA including 41S, 30S, 21S, and 18S-Eintermediate form. 5′ETS probe detects 45S primary transcript and 30Sintermediate form of mature 18S rRNA.

FIGS. 24A-24C show that depletion of LeuCAG3′tsRNA down-regulates RPS28protein level without affecting its mRNA level, whose depletion causesapoptosis. FIG. 24A shows a schematic picture of putative LeuCAG3′tsRNAand LNA CAGPM binding sites on 45S pre-rRNA. To identify the tsRNAbinding sites in 45S pre-rRNA, the RNAhybrid program was used andanalyzed with 18 bp, 20 bp, and 21 bp sequences from the 3′end of LeuCAGtRNA as a LeuCAG3′tsRNA, resulting in 5 putative binding sites that werepositioned in the 5′ETS, 1 site in ITS1, 1 site in ITS2, 3 sites in 28SrRNA, and 1 site in 3′ETS. Putative LeuCAG3′tsRNA binding site isindicated as a black bar. Putative LNA CAGPM binding site is indicatedas a red bar. FIG. 24B shows that LeuCAG3′tsRNA and LNA CAGPM does notbind to 45S pre-rRNA. To inhibit the interaction between LeuCAG3′tsRNAand 45S pre-rRNA, each designed LNA, based on the FIG. 24A result, wastransfected with HeLa cells for 24 hrs and Northern hybridization wasperformed. The sequences of each LNA are listed in Table 1. FIG. 24Cshows the normalized ribosomal RNA abundance of FIG. 17E. Since 21S and18S-E pre-rRNA are processed from 30S pre-rRNA, the signal from the 21Sand 18S-E pre-rRNA was normalized to the signal from each 30S pre-rRNAand was again normalized to each normalized value from the GL2-GFPcontrol transfection. The signal from the mature 18S rRNA could not bedirectly compared to the precursors because a different probe was used.

FIGS. 25A and 25B show that depletion of LeuCAG3′tsRNA suppresses RPS28translation in elongation phase. FIG. 25A shows that Harringtoninetreatment stalls RPS28 and GAPDH mRNA at 80S complex. The polysomalprofile was analyzed followed by treatment of harringtonine. Lysatepreparation, separation in sucrose gradient, and northern blots wereprocessed as in FIG. 18A. Harringtonine (−), only cycloximide treatmentshows RPS28 mRNA sedimentation across the gradient. Harringtonine,Harringtonine treatment shows that it blocks the first peptidyl transferand stalls mRNA at 80S complex. FIG. 25B shows the relative mRNAdistrivution of FIG. 18C. Upper graph shows RPS28 mRNA distributionafter treatment of indicated LNA with only cycloheximide (FIG. 18C,top). Bottom graph shows RPS28 mRNA distribution after treatment ofindicated LNA and harringtonine (FIG. 18C, bottom). Amounts of mRNAs foreach fraction were normalized using the sum of the mRNA across allfractions. X-axis is the fraction number; y-axis is % of mRNA abundance.

FIG. 26 is the list of significant differential gene expression fromFIG. 15.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

In various embodiments, the invention described herein featurescompositions and methods that down regulate tRNA-derived small RNAs(tsRNAs). Accordingly, the present invention provides compositions andmethods directed to oligonucleotides that are complementary to tsRNAs.In some embodiments, the tsRNA is leuCAG3tsRNA, which is derived fromthe mature leuCAG tRNA.

Surprisingly, the down regulation of tsRNAs, in particular leuCAG3tsRNA,by oligonucleotides of the present invention that employ differentinhibitory mechanisms, results in the induction of apoptosis. Thecompositions and methods accordingly provide a new approach to causingcell death and offer new treatments for disease, for example, cancer,autoimmune disease, overgrowth of non-malignant states, and excessivevascular states.

Definitions

As used herein, “tRNA-derived small RNAs” or “tsRNA” or tsRNAs” refersto small RNAs that are derived from the cleavage of, and there “sense”to, a tRNA.

As used herein, “leuCAGtsRNA” refers to a tsRNA derived from leuCAGtRNA; “leuCAG3tsRNA” refers to a tsRNA derived from the 3′ end of leuCAGtRNA, and “leuCAG5tsRNA” refers to a tsRNA derived from the 5′ end ofleuCAG tRNA.

As used herein, “tRNA” or “transfer RNA” refers to an RNA molecule thatserves as the physical link between the nucleotide sequence of nucleicacids and the amino acid sequence of proteins.

As used herein, the term “target tsRNA” encompasses tsRNA derived from atRNA molecule. The specific hybridization of an oligomeric compound withits target tsRNA interferes with the normal function of the tsRNA. Thismodulation of function of a tsRNA by compounds, which specificallyhybridize to it, is generally referred to as “complementary” or“antisense.”

As used herein, “oligonucleotide” refers to a single stranded nucleicacid molecule. An oligonucleotide can comprise ribonucleotides,deoxyribonucleotides, modified nucleotides (e.g. nucleotides with 2′modification, 3′ modifications, synthetic base analogs, locked nucleicacids, etc.), or combinations thereof. Modified oligonucleotides arepreferred in some embodiments over native forms having unmodifiednucleotides because of properties including, for example, enhancedbinding properties, increased stability in the presence of nucleases,and enhanced cellular uptake.

As used herein, “complementary oligonucleotides” or “antisenseoligonucleotides” or “oligonucleotide that is complementary” refers toan RNA or DNA molecule that binds to another RNA or DNA (target RNA,DNA). For example, if it is an RNA oligonucleotide it binds to anotherRNA target by means of RNA-RNA interactions and alters the activity ofthe target RNA. A complementary oligonucleotide can upregulate ordownregulate expression and/or function of a particular polynucleotide.The definition is meant to include any foreign RNA or DNA molecule whichis useful from a therapeutic, diagnostic, or other viewpoint.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, and complements thereof. The term refers to allforms of nucleic acids (e.g., gene, pre-mRNA, mRNA, tRNA) and theirpolymorphic variants, alleles, mutants, and interspecies homologs. Theterm nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide. The term encompasses nucleic acidsthat are naturally occurring or recombinant. Nucleic acids can (1) codefor an amino acid sequence that has greater than about 60% amino acidsequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of at least about 25, 50, 100, 200,500, 1000, or more amino acids, to a polypeptide encoded by a referencednucleic acid or an amino acid sequence described herein; (2)specifically bind to antibodies, e.g., polyclonal antibodies, raisedagainst an immunogen comprising a referenced amino acid sequence,immunogenic fragments thereof, and conservatively modified variantsthereof; (3) specifically hybridize under stringent hybridizationconditions to a nucleic acid encoding a referenced amino acid sequence,and conservatively modified variants thereof; (4) have a nucleic acidsequence that has greater than about 95%, preferably greater than about96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferablyover a region of at least about 25, 50, 100, 200, 500, 1000, or morenucleotides, to a reference nucleic acid sequence.

A particular nucleic acid sequence also implicitly encompasses “splicevariants” and nucleic acid sequences encoding truncated forms of aprotein. Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant ortruncated form of that nucleic acid. “Splice variants,” as the namesuggests, are products of alternative splicing of a gene. Aftertranscription, an initial nucleic acid transcript may be spliced suchthat different (alternate) nucleic acid splice products encode differentpolypeptides. Mechanisms for the production of splice variants vary, butinclude alternate splicing of exons. Alternate polypeptides derived fromthe same nucleic acid by read-through transcription are also encompassedby this definition. Any products of a splicing reaction, includingrecombinant forms of the splice products, are included in thisdefinition. Nucleic acids can be truncated at the 5′ end or at the 3′end. Polypeptides can be truncated at the N terminal end or theC-terminal end. Truncated versions of nucleic acid or polypeptidesequences can be naturally occurring or recombinantly created.

“Modified nucleic acids” can contain known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,locked nucleic acids (LNAs), and peptide-nucleic acids (PNAs). Modifiedbases include nucleotide bases such as, for example, adenine, guanine,cytosine, thymine, uracil, xanthine, inosine, and queuosine that havebeen modified by the replacement or addition of one or more atoms orgroups. Some examples of types of modifications that can comprisenucleotides that are modified with respect to the base moieties includebut are not limited to, alkylated, halogenated, thiolated, aminated,amidated, or acetylated bases, individually or in combination. Morespecific examples include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose,and other sugars, heterocycles, or carbocycles. An unmodified nucleicacid as used herein refers to DNA.

As used herein, “locked nucleic acid” or “LNA” refers to a modifiednucleotide where the ribose moiety is modified with an extra bridgeconnecting the 2′ oxygen and 4′ carbon. See, e.g. Koshkin et al.Tetrahedron 54:3607-30 (1998).

The term “identical” or “identity” or “percent identity,” or “sequenceidentity” in the context of two or more nucleic acids or polypeptidesequences that correspond to each other refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same (e.g., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region,when compared and aligned for maximum correspondence over a comparisonwindow or designated region) as measured using a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection. Such sequences are thensaid to be “substantially identical” and are embraced by the term“substantially identical.” This definition also refers to, or can beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists for a specified entire sequence or a specified portion thereof orover a region of the sequence that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. A corresponding region is anyregion within the reference sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters. A comparison windowincludes reference to a segment of any one of the number of contiguouspositions selected from the group consisting of from 20 to 600, usuallyabout 50 to about 200, more usually about 100 to about 150 in which asequence can be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted(e.g., by the local homology algorithm of Smith & Waterman, Adv. AppLMath. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds. 1995 supplement)).

An exemplary algorithm suitable for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402(1977) and Altschul et al., J Mol. Biol. 215:403-410 (1990),respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

As used herein, “apoptosis” or “programmed cell death” is thephysiological process for the killing and removal of unwanted cells andthe mechanism whereby chemotherapeutic agents kill cancer cells.Apoptosis is characterized by distinctive morphological changes withincells that include condensation of nuclear chromatin, cell shrinkage,nuclear disintegration, plasma membrane blebbing, and the formation ofmembrane-bound apoptotic bodies (Wyllie et al., Int. Rev. Cytol., 68:251, 1980). The translocation of phosphatidylserine from the inner faceof the plasma membrane to the outer face coincides with chromatincondensation and is regarded as a cellular hallmark of apoptosis(Koopman, G. et al., Blood, 84:1415, 1994). The actual mechanism ofapoptosis is known to be mediated by the activation of a family ofcysteine proteases, known as caspases.

As used herein, “cell necrosis” or “necrosis” refers to a form oftraumatic cell death that results from acute cellular injury.

As used herein “cell proliferation” refers to cellular growth.

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pincaloma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, gastric cancer, premalignant skin lesions, non-malignantstates such as warts or nevi, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, cervical cancer, endometrial cancer, adrenalcortical cancer, and prostate cancer. A nonmalignant state is anon-cancerous state also referred to as benign tumors. Nonmalignanttumors can grow but do not spread to other parts of the body.

As used herein, an “autoimmune disorder” refers to a disease or disorderthat arises from an inappropriate immune response against antigens thatare present and functionally normally within an organism. Non-limitingexamples of autoimmune disorders are Addison's disease, Amyotrophiclateral sclerosis, Atopic allergy, Cushing's Syndrome, Diabetes mellitustype 1, Eczema, Endometriosis. Lupus erythematosus, multiple sclerosis,and rheumatoid arthritis.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, peptide,circular peptide, lipid, fatty acid, siRNA, polynucleotide,oligonucleotide, etc., to be tested for the capacity to directly orindirectly modulate cancer biomarkers. The test compound can be in theform of a library of test compounds, such as a combinatorial orrandomized library that provides a sufficient range of diversity. Testcompounds are optionally linked to a fusion partner, e.g., targetingcompounds, rescue compounds, dimerization compounds, stabilizingcompounds, addressable compounds, and other functional moieties.Conventionally, new chemical entities with useful properties aregenerated by identifying a test compound (called a “lead compound”) withsome desirable property or activity, e.g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis. Compounds can be inhibitors,activators, or modulators of a tsRNA, and are used to refer toactivating, inhibitory, or modulating molecules identified using invitro and in vivo assays of the tsRNA. Inhibitors are compounds that,e.g., bind to, partially or totally block activity, decrease, prevent,delay activation, inactivate, desensitize, or down regulate the activityor expression of a tsRNA, e.g., antagonists. Activators are compoundsthat increase, open, activate, facilitate, enhance activation,sensitize, agonize, or up regulate tsRNA activity, e.g., agonists.

As used herein, the term “pharmaceutically acceptable carrier” refers tovarious solvents as described herein which can be employed in thepreparation of the formulations of the present invention.

As used herein, “patient” or “subject” refers broadly to any animal whois in need of treatment either to alleviate a disease state or toprevent the occurrence or reoccurrence of a disease state. Also,“Patient” as used herein, refers broadly to any animal who has riskfactors, a history of disease, susceptibility, symptoms, signs, waspreviously diagnosed, is at risk for, or is a member of a patientpopulation for a disease. The patient may be a clinical patient such asa human or a veterinary patient such as a companion, domesticated,livestock, exotic, or zoo animal. The term “subject” may be usedinterchangeably with the term “patient.” In preferred embodiments, apatient is a human.

The term “treating” (or “treat” or “treatment”) means slowing,interrupting, arresting, controlling, stopping, reducing, or reversingthe progression or severity of a symptom, disorder, condition, ordisease, but does not necessarily involve a total elimination of alldisease-related symptoms, conditions, or disorders. The terms“treating,” “treatment,” and “therapy” as used herein refer to curativetherapy, prophylactic therapy, and preventative therapy.

In certain embodiments, the therapy is designed to inhibit thedevelopment or progression of apoptotic diseases. The term “inhibit”means the ability to substantially antagonize, prohibit, prevent,restrain, slow, disrupt, eliminate, stop, reduce, or reverse thebiological effects of leuCAGtsRNA.

The term “effective amount” refers to the amount or dose of a compoundof the present invention which, upon single or multiple doseadministration to a patient, provides the desired treatment orprevention. A therapeutically effective amount for any individualpatient can be determined by the health care provider by monitoring theeffect of the compound(s) on a biomarker, such as a cell cycle,inflammation, apoptotic, or cancer biomarker. Analysis of the dataobtained by these methods permits modification of the treatment regimenduring therapy so that optimal amounts of compound(s), whether employedalone or in combination with one another therapeutic agent, areadministered, and so that the duration of treatment can be determined aswell. In this way, the dosing/treatment regimen can be modified over thecourse of therapy so that the lowest amounts of compound(s) used aloneor in combination that exhibit satisfactory tumor reducing effectivenessare administered, and so that administration of such compounds iscontinued only so long as is necessary to successfully treat thepatient.

In certain embodiments, the therapy is designed to treat developmentaldefects (Xue and Barna, 2012), malignant processes (Bywater et al.,2013), Treacher Collins syndrome (TCS), Shwachman-Bodian-Diamondsyndrome (SBDS), Dyskeratosis congenita, 5q⁻ syndrome, and DiamondBlackfan anemia (DBA) (reviewed in (Freed et al., 2010). In ceratinembodiments, the therapy is direct at diseases associated with and/orcaused by apoptotic defects. In ceratin embodiments, the therapy isdirect at diseases associated with and/or caused by ribosome biogenesisdefects. In certain embodiments, the therapy is cancer therapy. Incertain embodiments the therapy is used to treat an autoimmune disease,a non-maligant state, or an excessive vascular state. In certainembodiments, the therapy is used to treat hyperplasia or maculardegeneration.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer”, “cancerous”, and “tumor” are notmutually exclusive as used herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byaberrant cell growth/proliferation. Examples of cancers include, but arenot limited to, carcinomas, lymphomas, blastomas, sarcomas, andleukemias. The terms “cancer” and “cancerous” further refer to ordescribe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, renal cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals,nonhuman primates, and zoo, sports, or pet animals, such as dogs,horses, cats, cows, etc.

In certain embodiments wherein the compound(s) of the invention are usedin cancer therapy, they are used in combination with one or moreadditional anti-cancer agents. In certain embodiments, the anti-canceragent is a chemotherapeutic agent.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of conditions like cancer. Examples of chemotherapeutic agentsinclude alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin (₁ ^(I) and calicheamicin 2^(I)₁, see, e.g., Angew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folicacid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, 5-FU; androgens such ascalusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

In certain embodiments wherein the compound(s) of the invention are usedin a therapy (e.g., cancer therapy, proliferative disease therapy, oranti-inflammatory therapy), they are used in combination with one ormore additional anti-inflammatory agents. In certain embodiments, theanti-inflammatory agents are selected from a group comprisingsufasalazine, mesalamine, NSAIDs, ImSAIDs, and corticosteroids.

In certain embodiments wherein the compound(s) of the invention are usedin a therapy (e.g., cancer therapy, proliferative disease therapy, oranti-inflammatory therapy), they are used in combination with one ormore additional antibiotic (i.e., antibacterial) agents.

tRNA-Derived Small RNAs

The present invention provides oligonucleotides complementary totRNA-derived small RNAs (tsRNAs). A tsRNA can be any member of anon-coding regulatory RNA derived from tRNA. A tsRNA is thus usually“sense” to the tRNA from which it is derived. A tsRNA is usually 20-30nucleotides in length, but can be shorter in length, e.g. 15nucleotides, 10 nucleotides, or even 5 nucleotides or less. A tsRNAs canbe longer in length, e.g. 35 nucleotides, 40 nucleotides, 45nucleotides, or 50 or more nucleotides in length.

tsRNAs of the present invention can be referred to by any knownphraseology used in the art. For example, tsRNAs can include tRNAhalves, which are usually 28-36 nucleotides in length and are mainlygenerated by anticodon nucleases in bacteria (Ogawa et al. Science283:2097-2100 (1999)), Rnylp in yeast (Thompson and Parker, Cell138:215-219 (2009), and angiogenin in humans (Fu et al. FEBS Lett.583:437-442 (2009). tsRNAs of the present invention can also includetRNA fragments (tRFs), which are usually 14-22 nucleotides in length(Lee et al., Genes. Dev. 23 :2639-2649 (2009)). A tsRNA can be a5′tsRNA, and 3′ tsRNA (type I), or a tsRNA derived from the 3′ end of atRNA precursor.

Accordingly, tsRNAs can be any cleaved sequence of nucleotide derivedfrom a tRNA. In an embodiment, the tsRNA can be any tsRNA derived fromleuCAG tRNA, including but not limited to, leuCAG3tsRNA, leuCAG5tsRNA,and leuCAG3tsRNA. In other embodiments, the tsRNA is derived from aspGTCtRNA, serGCT tRNA, and metI tRNA.

A tsRNA can be derived from any organism, including but not limited to,E. coli, T. thermophila, S. coelicor, A. fumigatus, S. cerevisiae, G.lamblia, T. cruzi, D. melanogaster, A. thaliana, C. maxima, and anymammalian cell. A tsRNA can have any endogenous function as known in theart, including but not limited to, gene silencing; phage infection;reduction of uncharged tRNA; cell cycle progression; essential aminoacid regulation; cell differentiation; conidiogenesis; down-regulationof protein synthesis; encystation; stress maintenance includingoxidative stress, heat shock, UV hyperosmolarity, nutritional stress;and translational inhibition.

The tsRNAs of the present invention can be post-translationally modifiedeither endogenously within a cell, or in vitro. Such modification caninclude any known post-translational modification, for example,methylation.

tsRNA Oligonucleotides

In an embodiment, oligonucleotides are provided against tsRNAs. Theoligonucleotides can be complementary, antisense, orreverse-complementary to both a tsRNA derived from a tRNA, or the tRNAitself, which are terms known in the art. In an embodiment, theoligonucleotides are complementary to a tsRNA.

In some embodiments the oligonucleotide has a length of 7-25(contiguous) nucleotides, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 (contiguous) nucleotides. In someembodiments, the oligonucleotide is fully complementary to asub-sequence of contiguous nucleotides present in the tsRNA target. Insome embodiments, the oligonucleotide comprises one or more mismatchwith the complement of a sub-sequence of contiguous nucleotides presentin said tsRNA target.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligonucleotide can have 1, 2, 3, 4,5, or more mismatches. Thus, the oligonucleotides of the presentinvention can comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an oligonucleotide that is 18 nucleotides inlength having 4 (four) noncomplementary nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the an.Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).Methods of producing oligonucleotides are well known in the art, and canutilize, for example, an Expedite 8900/MOSS synthesizer (MultipleOligonucleotide Synthesis System). LNA monomer building blocks andderivatives can be prepared by any method well known in the art, forexample, as in WO 03/095467.

As such the oligonucleotide is an antisense oligonucleotide in that itis either fully complementary to the (corresponding) target sequence, orcomprises one or more mismatches with the target sequence.

In some embodiments, the oligonucleotides are mixmers, which compriseboth naturally occurring unmodified, and non-naturally occurringnucleotides (e.g. locked nucleic acids (LNAs)), where, as opposed togapmers, tailmers, headmers and blockmers, there is no contiguoussequence of unmodified naturally occurring nucleotides, such as DNAunits. As known in the art, mixmers effectively and specifically bind totheir target, and the use of mixmers as therapeutic oligomers areconsidered to be particularly effective in decreasing the target RNA. Insome embodiments the oligonucleotide can be a totalmer, which onlycomprise modified nucleotides. In some embodiments, the oligonucleotidecan be a gapmer, which is a series of contiguous unmodified nucleicacids flanked on both sides by a series of contiguous nucleotides thatare modified, such as with LNAs.

In some embodiments, the oligonucleotide binds to a target tsRNA andcleaves the target by RNAse mediated degradation. In some embodiments,the oligonucleotide binds to a target tsRNA and prevents the tsRNA fromassociating with any other molecule.

In some embodiments, the mixmer comprises or consists of a contiguousnucleotide sequence of repeating pattern of nucleotide analogue andnaturally occurring nucleotides, or one type of modified nucleotideanalogue and a second type of modified nucleotide analogues. Therepeating pattern, may, for instance be every second or every thirdnucleotide is a nucleotide analogue, such as LNA, and the remainingnucleotides are naturally occurring nucleotides, such as DNA. It isrecognized that the repeating pattern of nucleotide analogues, such asLNA units, may be combined with nucleotide analogues at fixed positionse.g. at the 5′ or 3′ termini.

The oligonucleotide of claim 1, wherein the oligonucleotide is of astructure according to Formula (I).(A)_(x)-(B)_(y)-(C)_(z)  Formula (I)

In Structure 1, x, y, and z are integers that are greater than or equalto 1. A is a modified or unmodified nucleic acid. When x is greater than1, each A is independently selected and A is the 5′ end of theoligonucleotide. B is a modified or unmodified nucleic acid. When y isgreater than 1, each B is independently selected. C is a modified orunmodified nucleic acid. When z is greater than 1, each C isindependently selected and C is the 3′ end of the oligonucleotide.

Accordingly, an embodiment of Structure 1 can include both mixmer andgapmer oligonucleotides. For example, when A is either a single modifiednucleic acid, or A comprises two or more nucleic acids that are each amodified nucleic acid; and when B is either a single unmodified nucleicacid, or B comprises two or more nucleic acids that are each unmodifiednucleic acids; and when C is either a single modified nucleic acid, or Ccomprises two or more nucleic acids that are each modified nucleicacids; then the oligonucleotide is a gapmer. In contrast, when each A isindependently selected as either a modified or unmodified nucleic acid,when each B is independently selected as either a modified or unmodifiednucleic acid, when each C is independently selected as either a modifiedor unmodified nucleic acid, and when at least one of A, B, or Ccomprises at least one modified and one unmodified nucleic acid, thenthe oligonucleotide is a mixmer.

In some embodiments, the first 5′ nucleotide of the oligonucleotide ismodified, for example, is LNA. In some embodiments, the first fournucleotides of the oligonucleotide are modified, for example, are LNA.In some embodiments, the last nucleotide of the oligonucleotide ismodified, for example, is LNA. In some embodiments, the last fournucleotides of the oligonucleotides are modified, for example, are LNA.In some embodiments, a gapmer oligonucleotide has 10 unmodifiednucleotides (e.g. is B of Structure 1). In some embodiments, the second,third, fifth, sixth, eighth, tenth, thirteenth, and fifteenth nucleotidefrom the 5′ end of a 15 nucleotide mixmer oligonucleotide is modified,e.g. is LNA, and the first, fourth, seventh, ninth, eleventh, twelfth,and fourteenth nucleotide from the 5′ end of a 15 nucleotide mixeroligonucleotide is unmodified, e.g., is DNA.

Exemplary oligonucleotides of the present invention are the mixmeroligonucleotide 5′ tGTcAGgAgTggGaT 3′ (SEQ ID NO: 2) and the gapmeroligonucleotide 5′ GGTGtcaggagtggGATT 3′ (SEQ ID NO: 11) whereinuppercase letters represent a modified nucleic acid, e.g. LNA, andlowercase letters represent unmodified nucleic acids, e.g. DNA.Additional oligonucleotides that are exemplary to the present inventionare provided in Table 5 and Table 6.

In an embodiment, the modified nucleic acid can be any modified nucleicacid known in the art, which can include, but is not limited to, a2′-O-alkyl-RNA unit, a 2′-OMe-RNA unit, a 2′-amino-DNA unit, a2′-fluoro-DNA unit, a 2′-MOE-RNA unit, a LNA unit, a PNA unit, a HNAunit, or an INA unit.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarily to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

Pharmaceutical Compositions

In various embodiments, a pharmaceutical formula comprises anoligonucleotide of the present invention in a pharmaceuticallyacceptable carrier. Appropriate pharmaceutically acceptable carriers aredetermined in part by the particular composition being administered(e.g., the oligonucleotides comprising modified nucleic acids asdescribed herein), as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed.,1989).

Pharmaceutical formulations within the scope of the present inventioncan also contain other compounds, which can be biologically active orinactive. Pharmaceutical compositions can generally be used forprophylactic and therapeutic purposes.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the nucleic acid diluted in adiluent, such as water, saline or PEG 400; (b) capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (e.g., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, for example, byintraarticular (in the joints), intravenous, intramuscular, intradermal,intraperitoneal, and subcutaneous routes, can include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials. Methods for preparing suchdosage forms are understood by those skilled in the art (see, e.g.,REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Ed., Mack Publishing Co.,Easton, Pa. (1990)).

Pharmaceutical compositions can also comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, bacteriostats, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminumhydroxide), solutes that render the formulation isotonic, hypotonic orweakly hypertonic with the blood of a recipient, suspending agents,thickening agents and/or preservatives. Alternatively, compositions ofthe present invention can be formulated as a lyophilizate. Compounds canalso be encapsulated within liposomes using well known technology.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

Inducing Apoptosis

In various embodiments, the invention provides a method to induceapoptosis or necrosis via the specific down regulation of a tsRNA, e.g.,leuCAG3tsRNA (FIGS. 8-10).

The compositions and methods accordingly provide a new approach tocausing cell death and offer new treatments for diseases caused byabnormalities in the control of apoptosis, which can result in either apathological increase in cell number (e.g. cancer) or a damaging loss ofcells (e.g. degenerative diseases). As non-limiting examples, themethods and compositions of the present invention can be used to preventor treat a subject having the following apoptosis-associated diseasesand disorders. Exemplary disorders includeneurological/neurodegenerative disorders (e.g., Alzheimer's,Parkinson's, Huntington's, Amyotrophic Lateral Sclerosis (Lou Gehrig'sDisease), autoimmune disorders (e.g., rheumatoid arthritis, systemiclupus erythematosus (SLE), multiple sclerosis), Duchenne MuscularDystrophy (DMD), motor neuron disorders, ischemia, heart ischemia,chronic heart failure, stroke, infantile spinal muscular atrophy,cardiac arrest, renal failure, atopic dermatitis, sepsis and septicshock, AIDS, hepatitis, glaucoma, diabetes (type 1 and type 2), asthma,retinitis pigmentosa, osteoporosis, xenograft rejection, and burninjury.

Once made, the compositions of the invention find use in inhibiting cellviability. In an embodiment, viability of a cell is inhibited byadministering to the cell the oligonucleotides of the present invention.In an embodiment, inhibiting a tsRNA inhibits the viability of a cell.In an embodiment, inhibiting a tsRNA induces apoptosis. In anembodiment, inhibiting leuCAG3tsRNA induces the viability of a cell. Inan embodiment, inhibiting leuCAG3tsRNA induces apoptosis.

In an embodiment, the viability of a cell is inhibited by administeringto the cell an oligonucleotide as described herein. One of skill in theart would understand that the oligonucleotides described herein functionas mixmers with modified nucleic acids such as LNA, as gapmers withmodified nucleic acids such as LNA, or oligonucleotides with unmodifiednucleic acids, such as DNA. Accordingly, one of skill in the art wouldunderstand that an oligonucleotide can inhibit its target by more thanmode of action, e.g. by RNAase degradation wherein the oligonucleotidebinds to a target tsRNA and cleaves the target; or by binding to atarget tsRNA and preventing the tsRNA from associating with any othermolecule.

In an embodiment, the viability of a cell is inhibited, or apoptosis isinduced, by administering to a cell any compound or modulator of atsRNA. In an embodiment, the viability of a cell inhibited, or apoptosisis induced, by administering to a cell any compound or modulator ofleuCAG3tsRNA.

Modulation of a tsRNA such as leuCAG3tsRNA can be assessed using avariety of in vitro and in vivo assays, including cell-based models asdescribed herein. Such assays can be used to test for inhibitors andactivators of a tsRNA such as leuCAG3tsRNA.

Assays to identify compounds with tsRNA-modulating activity, such asleuCAG3tsRNA, can be performed in vitro. In an exemplary assay, thetsRNA is bound to a solid support, either covalently or non-covalently.Often, the in vitro assays of the invention are substrate or ligandbinding or affinity assays, either non-competitive or competitive. Otherin vitro assays include measuring changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the tsRNA.

A high throughput binding assay can be performed in which the tsRNAthereof is contacted with a potential modulator and incubated for asuitable amount of time. In one embodiment, the potential modulator isbound to a solid support, and the tsRNA is added. A wide variety ofmodulators can be used, as described below, including small organicmolecules, peptides, antibodies, etc.

In an embodiment, high throughput screening methods involve providing acombinatorial small organic molecule or peptide library containing alarge number of potential therapeutic compounds (potential modulator orligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(e.g., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., I Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., I Org. Chem. 59:658 (1994)), nucleic acidlibraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleicacid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries(see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996)and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C & E N,Jan. 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

Methods of Treatment

The compositions of the invention find use in methods of treatment. Inan embodiment, the present invention provides methods of treating adisease in a subject. The oligonucleotide or pharmaceutical formulathereof, is typically administered to the subject in a therapeuticallyeffective dose, which may for example be determined by a dose which issufficient to down-regulate the target tsRNA, or activity thereof, to asignificant level over the time period between successive administrationdosages, such as a level which is a therapeutic benefit to the subject.In some embodiments, the target tsRNA, or activity thereof isdown-regulated by at least 10%, such as at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70% or at least 80% orat least 90% during the time period between successive administrationdosages. In an embodiment, the method of treatment includes inhibitingthe function of a tsRNA. In an embodiment, the tsRNA is leuCAG3tsRNA.The compositions can include any oligonucleotide described herein,including those with and without modified nucleic acids.

Administration of the compositions of the present invention with asuitable pharmaceutical excipient can be carried out via any acceptedmodes of administration. Thus, administration can be, for example,intravenous, topical, subcutaneous, transcutaneous, transdermal,intramuscular, oral, intra joint, parenteral, intra-arteriole,intradermal, intraventricular, intracranial, intraperitoneal,intralesional, intranasal, rectal, vaginal, or by inhalation.Administration can be targeted directly to pancreatic tissue, e.g., viainjection.

The compositions of the invention may be administered repeatedly, e.g.,at least 2, 3, 4, 5, 6, 7, 8, or more times, or the composition may beadministered by continuous infusion. Suitable sites of administrationinclude, but are not limited to, dermal, mucosal, bronchial,gastrointestinal, anal, vaginal, and ocular. The formulations may takethe form of solid, semi-solid, lyophilized powder, or liquid dosageforms, such as, for example, tablets, pills, lozenges, capsules,powders, solutions, suspensions, emulsions, suppositories, retentionenemas, creams, ointments, lotions, gels, aerosols, or the like,preferably in unit dosage forms suitable for simple administration ofprecise dosages.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient. In some embodiments, the dosage of the compoundadministered at each dosing, such as unit dose, is within the range ofabout 0.01 mg/kg to about 25 mg/kg. In some embodiments, the dosage,such as unit dose, of the compound administered at each dosing is withinthe range of about 0.05 mg/kg to about 20 mg/kg. In some embodiments,the dosage (such as unit dose) of the compound administered at eachdosing is within the range of about 0.1 mg/kg to about 15 mg/kg. In someembodiments, the (such as unit dose) dosage of compound administered ateach dosing is within the range of about 1 mg/kg to about 15 mg/kg. Insome embodiments, the dosage of the compound administered at each dosingis within the range of about 1 mg/kg to about 10 mg/kg. In someembodiments, the dosage (such as unit dose) of the compound administeredat each dosing is within the range of about 0.01 mg/kg to about 25mg/kg, such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4,4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5,7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75,11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25, 13.5, 13.75,14, 14.25, 14.5, 14.75, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or suchas about 25 mg/kg, each of which are individual embodiments.

In another approach to treatment, modified and unmodified nucleic acidscan be used for transfection of cells in vitro and in vivo. Thesenucleic acids can be inserted into any of a number of well-known vectorsfor the transfection of target cells and organisms as described below.The nucleic acids are transfected into cells, ex vivo or in vivo,through the interaction of the vector and the target cell. The nucleicacids, under the control of a promoter, then express a polypeptide ofthe present invention, thereby mitigating the effects of a diseaseassociated with reduced insulin production.

Embodiments of the present invention can be used to treat sarcomas andcarcinomas including, but not limited to: fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pincaloma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, gastric cancer, premalignant skin lesions, non-malignantstates such as warts or nevi, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, cervical cancer, endometrial cancer, adrenalcortical cancer, and prostate cancer.

Embodiments of the present invention can be used to treat diseasesassociated with mis-regulation of apoptosis including, but not limitedto neurological/neurodegenerative disorders (e.g., Alzheimer's,Parkinson's, Huntington's, Amyotrophic Lateral Sclerosis (Lou Gehrig'sDisease), autoimmune disorders (e.g., rheumatoid arthritis, systemiclupus erythematosus (SLE), multiple sclerosis), Duchenne MuscularDystrophy (DMD), motor neuron disorders, ischemia, heart ischemia,chronic heart failure, stroke, infantile spinal muscular atrophy,cardiac arrest, renal failure, atopic dermatitis, sepsis and septicshock, AIDS, hepatitis, glaucoma, diabetes (type 1 and type 2), asthma,retinitis pigmentosa, osteoporosis, xenograft rejection, and burninjury.

The methods and compositions of the present invention have use as genetherapy treatments. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies (for a review of gene therapy procedures, see Anderson,Science, 256:808-813 (1992); Nabel et al., TIBTECH, 11:211-217 (1993);Mitani et al., TIBTECH, 11:162-166 (1993); Mulligan, Science, 926-932(1993); Dillon, TIBTECH, 11:167-175 (1993); Miller, Nature, 357:455-460(1992); Van Brunt, Biotechnology, 6(10):1149-1154 (1998); Vigne,Restorative Neurology and Neuroscience, 8:35-36 (1995); Kremer et al.,British Medical Bulletin, 51(1):31-44 (1995); Haddada et al., in CurrentTopics in Microbiology and Immunology (Doerfler & Bohm eds., 1995); andYu et al., Gene Therapy, 1:13-26 (1994)).

For delivery of nucleic acids, including oligonucleotides comprisingmodified nucleic acids such as locked nucleic acids, or oligonucleotidescomprising only unmodified nucleic acids, viral vectors may be used.Suitable vectors include, for example, herpes simplex virus vectors asdescribed in Lilley etal., Curr. Gene Ther 1(4):339-58 (2001),alphavirus DNA and particle replicons as described in e.g., Polo et al.,Dev. Biol. (Basel), 104:181-5 (2000), Epstein-Barr virus (EBV)-basedplasmid vectors as described in, e.g., Mazda, Curr. Gene Ther.,2(3):379-92 (2002), EBV replicon vector systems as described in e.g.,Otomo et al., J. Gene Med., 3(4):345-52 (2001), adeno-virus associatedviruses from rhesus monkeys as described in e.g., Gao et al., PNAS USA.,99(18):11854 (2002), adenoviral and adeno-associated viral vectors asdescribed in, e.g., Nicklin et al., Curr. Gene Ther 2(3):273-93 (2002).Other suitable adeno-associated virus (AAV) vector systems can bereadily constructed using techniques well known in the art (see, e.g.,U.S. Pat. Nos. 5,173,414 and 5,139,941; PCT Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 (1988);Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, Current Opinion in Biotechnology 3:533-539 (1992); Muzyczka,Current Topics in Microbiol. and Immunol., 158:97-129 (1992); Kotin,Human Gene Therapy, 5:793-801 (1994); Shelling etal., Gene Therapy,1:165-169 (1994); and Zhou et al., J. Exp. Med., 179:1867-1875 (1994)).Additional suitable vectors include E1B gene-attenuated replicatingadenoviruses described in, e.g., Kim et al., Cancer Gene Ther9(9):725-36 (2002) and nonreplicating adenovirus vectors described ine.g., Pascual et al., J. Immunol., 160(9):4465-72 (1998). Exemplaryvectors can be constructed as disclosed by Okayama et al., Mol. Cell.Biol., 3:280 (1983).

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem., 268:6866-6869 (1993) andWagner et al., Proc. Natl. Acad. Sci. USA, 89:6099-6103 (1992), can alsobe used for gene delivery according to the methods of the invention.

Retroviruses can provide a convenient and effective platform for genedelivery systems. Retroviruses can be used to express theoligonucleotides of the present invention, including oligonucleotidescomprising modified nucleic acids such as locked nucleic acids, oroligonucleotides comprising only unmodified nucleic acids. A selectednucleotide sequence encoding a polypeptide of the invention is insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to a subject. Suitable vectors include lentiviral vectors asdescribed in e.g., Scherr et al., Curr. Gene Ther., 2(1):45-55 (2002).Additional illustrative retroviral systems have been described (e.g.,U.S. Pat. No. 5,219,740; Miller et al., BioTechniques, 7:980-990 (1989);Miller, Human Gene Therapy, 1:5-14 (1990); Scarpa et al., Virology,180:849-852 (1991); Burns et al., Proc. Natl. Acad. Sci. USA,90:8033-8037 (1993); and Boris-Lawrie et al., Curr. Opin. Genet.Develop., 3:102-109 (1993)).

Other known viral-based delivery systems are described in, e.g.,Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA, 86:317-321 (1989);Flexner et al., Ann. N.Y. Acad. Sci., 569:86-103 (1989); Flexner et al.,Vaccine, 8:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques, 6:616-627 (1988);Rosenfeld et al., Science, 252:431-434 (1991); Kolls et al., Proc. Natl.Acad. Sci. USA, 91:215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad.Sci. USA, 90:11498-11502 (1993); Guzman et al., Circulation,88:2838-2848 (1993); Guzman et al., Cir. Res., 73:1202-1207 (1993); andLotze et al., Cancer Gene Ther., 9(8):692-9 (2002).

EXAMPLES

The methods system herein described are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting.

Cell culture and transfection: Hela, HCT-116, and HEK293T cells weregrown in Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL®) with 2 mMof L-glutamine, and 10% heat-inactivated fetal bovine serum withantibiotics. All transfection assays were done with Lipofectamine 2000(Invitrogen®) according to the manufacturer's protocol. Locked nucleicacids (LNA) oligos were synthesized by Exiqon, which are listed onTable 1. 2′-o-methylated single strand RNAs were synthesized by IDT®(Integrated DNA Technologies), which are listed on Table 1. The siRNAswere purchased from Dharmacon® or Life Technologies®.

TABLE 1 The list of Locked nucleic acid (LNA) and 2′O-methylated singlestranded RNA, Related to FIG. 3 and 24. LNA bases are upper-case letter and DNA bases are lower-case letter. All bases of2′O-methylated single stranded RNA are 2′-o-methylated. LNA SequencesGL2c GtaCgCgGaaTaCTtC CAGPM tGTcAGgAgTggGaT CAGMM tCTcACgAgTggGaT CAGMM2tGTcAAgAcTggGaT CAG5′tsPM gcTcgGcCaTCcTgA CAGcodon GaGacTgcGAccTGa AspPMCCCgtcggggaatcgaACC SerPM cgACgAGgAtGggAt MetiPM TagCaGAgGAtgGTt GAP_GL2CGTAcgcggaatacTTCG GAP_5′tsPM GACCgctcggccatccTGAC GAP_3′tsPMGGTGtcaggagtggGATT GAP_codonPM GACTgcgacctgaacGCAG r571+CG + CA + CC + CC + ACG + CC + TTC + CC r1936C + C + CGA + CA + CG + CC + CG + C + AC + CA + C r2075+AT + CC + C + AC + CG + CC + AC + AGA + C + AC r2960a+CC + AC + CC + CC + CG + AC + C + CG r2960b+CC + CC + AC + CC + CC + CG + AC + CCG + G r5823aC + CG + CG + CC + CG + CCG + A + CA r5823b+CG + CC + CG + CCG + AC + A + CC + C + AC + GT r6162+AC + C + AC + CG + CCC + C + CG + AC r8527+CC + CA + CC + CC + CG + CA + CC + C r8546+CCG + A + CC + CC + AC + CC + C + CG r9079+CG + CC + CCG + CC + CC + CCG + A + C r12034+CG + CC + AG + A + AG + CG + AG + AG r4143+GG + TC + GG + GA + GT + GG + GT 2′-o-methylated single strand RNAControl UACGGACUUAAGCGGCUAC LeuCAG3′ts18mer AUCCCACUCCUGACACCALeuCAG3′ts2lmer UCGUAUCCCACUCCUGACACCA LeuCAG3′ts26merGGGUUCGAAUCCCACUCCUGACACCA

Dual-Luciferase Reporter Assay and cloning: 100 ng of pGL3 controlreporter plasmids and 20 ng of pRL reporter plasmids was transfectedwith 60 nM of LNA into Hela or HCT-116 cells in 24 well plates.FF-luciferase and RL-luciferase activities were measured 24 hr aftertransfection by using Promega's dual-luciferase kit protocol anddetected by a Modulus Microplate Luminometer (Turner BioSystems). Eachcomplementary sequences of tested small RNAs were cloned into 3′UTR or5′UTR of pGL3 control luciferase plasmid using XbaI and EcoRI or HindIIIand NcoI enzymes respectively. All complementary sequences of tsRNA,Let-7, and control sequences were obtained from dimerization of eachsense and anti-sense primers respectively, synthesized by IDT(Integrated DNA Technologies) (Table 2). PRDM10 5′UTR or 3′UTR wasamplified from HeLa cell line cDNA and cloned into 5′UTR or 3′UTR ofpGL3 control luciferase vector respectively (Table 3). The firefly gene,of which all CUG codons were replaced by CUC codons, from psicheck 2luciferase vector was synthesized from Life Technologies. All requiredprimers are listed on Table 3.

TABLE 2DNA oligonucleotides for target sequences in luciferase vector, Related to theExperimental Procedures. scramble S5′-ACACGTCGACGTATATAGCTCATTCatgcatACACGTCGACGT ATATAGCTCATTC-3′ (sense)scramble AS5′-GAATGAGCTATATACGTCGACGTGTatgcatGAATGAGCTATA TACGTCGACGTGT-3′(anti sense) LeuCAG3′tsPM S5′-TGGTGTCAGGAGTGGGATTCGAACCatgcatTGGTGTCAGGAG TGGGATTCGAACC-3′LeuCAG3′tsPM AS5′-GGTTCGAATCCCACTCCTGACACCAatgcatGGTTCGAATCCC ACTCCTGACACCAT-3′SerGCT3′tsPM S5′-TGGCGACGAGGATGGGATACGAACCCagtcTGGCGACGAGG ATGGGATACGAACCC-3′SerGCT3′tsPM AS5′-GGGTTCGTATCCCATCCTCGTCGCCAgactGGGTTCGTATCCC ATCCTCGTCGCCA-3′AspGTC3′tsPM S5′-TGGCTCCCCGTCGGGGAATCGAACCCCagtcTGGCTCCCCGT CGGGGAATCGAACCCC-3′AspGTC3′tsPM AS5′-GGGGTTCGATTCCCCGACGGGGAGCCAgactGGGGTTCGATT CCCCGACGGGGAGCCA-3′Let-7PM S 5′-CTAGAAACTATACAACCTACTTTTATAG-3′ Let-7PM AS5′-AATTCTGAGGTAGTAGGTTGTATAGTTT-3′

TABLE 3 Oligonucleotide for Northern probe. SerGCT5′tsaACCACTCGGCCACCTCGTC Northern probe SerGCT3′ts CGACGAGGGTGGGATTCGNorthern probe AspGTC3′ts CTCCCCGTCGGGGAATCG Northern probe leuCAG5′tsTAGACCGCTCGGCCATCCTGAC Northern probe leuCAG3′ts GTGTCAGGAGTGGGATTCGNorthern probe MetCAT I 3′ts GTAGCAGAGGATGGTTTCGA Northern probeMetCAT e 3′ts GTGCCCCGTGTGAGGATCGA Northern probe miR-15aacaaaccattatgtgctgcta Northern probe let-7 NorthernAACTATACAACCTACTACCTCA probe miR-92a gACAGGCCGGGACAAGTGCAATANorthern probe 28S Northern AACGATCAGAGTAGTGGTATTTCACC probe18S Northern CGGAACTACGACGGTATCTG probe ETS1 Northerngagagcacgacgtcaccacatcgatcacgaagagc probe 5′-ITS1gcctcgccctccgggctccgttaatgatc Northern probe ITS2b NortherngCTGCGAGGGAACCCCCAGCCGCGCA probe

RNA isolation and northern blotting: Hela and HCT-116 cells in 6 cmdishes were transfected with each 60 nM of LNA. Total RNA was isolatedat 24 h after transfection with TRIZOL reagent (Life Technologies®)according to the manufacturer's instructions. Total RNA was resolved byelectrophoresis on 15% (w/v) polyacrylamide gel with 7 M urea fordetection of small RNA whose size is smaller than 200 bp or on 0.9%agarose denaturating gel for detection of large RNA whose size is biggerthan 200 bp. After transfer onto Hybond-N+ nylon membrane (Amersham).P32-labeled oligonucleotide or amplified cDNA probes were hybridized tothe membrane in PerfectHyb™ Plus hybridization buffer (Sigma®). Alloligonucleotides for northern probes are listed on Table 3. Alloligonucleotides for cDNAs as northern probes are listed on Table 4.

TABLE 4 DNA oligonucleotides for PCR amplification,Related to the Experimental Procedures. PRDM10 5′UTR atgca GAGCTCforward AACATAGCAAGGTAGATATCAC PRDM10 5′UTR atgca GCTAGC reverseTTTAAACAGCTCAGGCAGGCTG PRDM10 3′UTR atgcaGATATCcttccaccctggagcttgaatcforward PRDM10 3′UTR atgcaCTGCAGgcttcacacatacaaacatg reverse NOP10_cDNAat gtttctccag tattacctc forward Nop10_cDNA tcagaggacagggcgcggttgcreverse RPS28_cDNA gccgcc atggacacc agccgtgtgc forward RPS28_cDNAtcagcgcaacctccgggcttc reverse RPS6_cDNAatgcat gatatc atgaagctgaacatctccttc forward RPS6_cDNAatgcat gaattc ttatttctgactggattcagac reverse RPS23_cDNAatgcat gatatc atgggca agtgtcgtgg ac forward RPS23_cDNAatgcat gaattc ttatgatcttggtctttccttc reverse RPS13_cDNATCGGCTTTACCCTATCGACG forward RPS13_cDNA CAAACGGTGAATCCGGCTCT reverse

Measurement of cell proliferation: Cell proliferation was measured withCellTiter 96 nonradioactive cell proliferation assay kit (MTT assay;Promega®) according to the manufacturer's instructions. All experimentswere performed in triplicates, from which average and standard deviationwere calculated and plotted.

Apoptosis assay: The cell apoptosis assay was performed by measuringtranslocation of membrane phospholipid phosphatidylserine using anAnnexin V-FITC apoptosis detection kit (BD Pharmingen®) according to themanufacturer's protocol. Cells were analyzed by FACScalibur instrumentusing FowJo software (Tree Star®). For the TUNEL assay, the apoptoticresponses were identified 24 h post-transfection using Invitrogen'sClick-iT® TUNEL Alexa Fluor® 594 Imaging Assay kit according to themanufacturer's protocol.

Western Blotting: 24 h post-transfection, cell lysates were preparedusing 1× cell lysis buffer (Cell Signaling) with 1 mM PMSF (CellSignaling®). 15 ug of protein lysate was run on 4-20% SDS PAGE andtransferred to Hybond-P membrane (GE Healthcare®). The membrane wasincubated for 20 min at room temperature (RT) in 4% BSA (Omnipur®)solution, washed, and incubated for over night (O/N) at 4° C. with oneof the following antibodies. After washing and incubation for 1 hr at RTwith secondary antibody, protein signal was detected using Pierce ECL2substrate (Thermoscientific®) according to manufacturer's protocol.Antibodies for immunoblotting were as follows: anti-PARP rabbitmonoclonal Ab (clone 46D11; Cell Signaling®), anti-cleaved PARP (Asp214)rabbit monoclonal Ab (clone D64E10; Cell Signaling®),anti-GAPDH-peroxidase mouse Ab (Sigma®), anti-RPS13 rabbit polyclonal Ab(Abnova®), anti-RPS7, 10, 16, 19, 24, and 28 Ab (Abcam®), RPL7, 13a, 26,and XRN2 Ab (Abcam®), anti-RPS6 mouse monoclonal Ab (Cell signal®).

High throughput large RNA sequencing: After transfection for 24 hrs,total RNA was isolated using TRIZOL reagent. Residual DNA contaminationand ribosomal sequences were removed from total RNA using TURBODNA-free™ Kit (Life technologies) and Rib o-Zero Gold™ kit (Epicentre)according to manufacturer's instructions respectively. A total of150-200 ng of purified RNA was subjected to strand-specific RNAseq usingthe ScriptSeq v2 mRNA-SEQ library preparation kit (Epicentre), whichuses a random-hexamer method of cDNA synthesis according tomanufacturer's instructions. 50 bp paired-end reads were generated on anIllumina HiSeq 2000 machine yielding a total of between 18 and 40million paired-end reads. Sequences were mapped to the human hg19 genomeusing TopHat version 1.4.1 with the following parameters: -r 180,-mate-std-dev 130 -library-type fr-secondstrand (Trapnell et al., 2009).FPKM values were calculated using the CuffDiff program version 1.3.0(Trapnell et al., 2012; 2010) using refGene transcripts as an input filefor comparison of gene expression levels.

Polysome gradient and RNA preparation: Polysome gradient and RNApreparation were performed as described previously (Fuchs et al., 2011)with modifications. After transfection for 24hrs, cells were treatedwith 100 jig/ml cycloheximide for 5 min, and cells were lysed in buffercontaining 15 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl2, 500 u/mlRNasin (Promega), 20 u/ml SUPERaseIn (Life Technologies) and 1% TritonX-100. For harringtonine treatment, after transfection for 24 hrs, cellswere treated with 2 ug/ml harringtonine for 2 min, followed by treatmentof 100 ug/ml cycloheximide for 5 min, and then lysed. The clearedlysates were loaded onto 10-50% sucrose gradients [15 mM Tris-HCl (pH7.5), 150 mM KCl, 5 mM MgCl2, 20 u/ml SUPERaseIn (Life Technologies),and 100 jig/ml cycloheximide] with a 60% sucrose cushion. Gradients werecentrifuged at 35,000 rpm at 4° C. for 2 hr 45 min and fractionated witha Teledyne Isco Foxy R1 Retriever/UA-6 detector system. The gradientfractions were sequentially treated for 30 min at 37° C. with 0.5 mg/mlproteinase K (New England Biolabs) in the presence of 5 mM EDTA. RNAswere extracted with an equal volume of phenol-chloroform-isoamylalcohol(25:24:1; Life Technologies). Aqueous phases were re-extracted withchloroform and precipitated with 3 M sodium acetate, 1 μl of 15 mg/mlglycogen (Life Technologies), 100% ethanol at −20° C. overnight, washedwith 75% ethanol, and resuspended in DEPC-water.

Puromycin gradients: Puromycin gradients were performed as describedpreviously (Fuchs et al., 2011) with modifications. After transfectionfor 24 hr, cells were lysed in buffer containing 15 mM Tris-HCl (pH7.5), 500 mM KCl, 2 mM MgCl2, 2 mM puromycin, 500 u/ml RNasin (Promega),20 u/ml SUPERaseIn (Life Technologies), and 1% Triton X-100. Followingincubation on ice for 15 min, 80S ribosome subunits were separated at37° C. for 10 min. Following centrifugation, cleared lysates were loadedonto 10-50% sucrose [500 mM KCl, 15 mM Tris-HCl (pH 7.5), 2 mM MgCl2,and 20 u/ml SUPERaseIn (Life Technologies)] with a 60% sucrose cushion.Gradients were centrifuged at 35,000 rpm at 4° C. for 2 hr 45 min. Ascontrol, cells were lysed and separated in gradients containing 15 mMTris-HCl (pH 7.5), 500 mM KCl, 15 mM MgCl2, 20 u/ml SUPERaseIn (LifeTechnologies), 100 ug/ml cycloheximide. The lysis buffer also contained1% Triton X-100.

Metabolic labeling of cell proteins with [³⁵S]-Methionine: Aftertransfection of indicated LNA for 24 hrs, cells were washed twice withPBS and were grown in DMEM media without Cystine and Methionine(DMEM-Cys-Met) (Life Technologies) for 30 min at 37° C. The media wasremoved and DMEM-Cys-Met plus 100 uCi protein labeling mix (PerkinElmer) was added for 10 min at 37° C. Cells were washed twice with coldPBS, harvested, and lysed with RIPA buffer. Equal amount of proteinswere resolved on 4-12% SDS PAGE, stained with coomasie brilliant blue,the gel was dried and the incorporated radioactivity was scanned usingPMI (Personal Molecular Imager).

Immunoprecipitation: Antibodies were incubated with protein A/GUltraLink Resin (Thermo Scientific) for o/n at 4° C. Cells were lysedwith IP buffer (25 mM Tris pH7.5, 150 mM Kcl, 0.5% NP40, 0.02 mM EDTA)containing RNasin® plus (promega) and cOmplete Protease InhibitorCocktail Tablets (Roche) and incubated with prepared protein A/Gconjugated Antibodies for 2 hrs at 4° C. All samples were washed with IPbuffer 3 times. Immunoprecipitated RNAs were extracted using TRIZOL(Life Technologies) according to the manufacturer's protocol.Immunoprecipitated proteins were separated from protein A/G UltraLinkresin by adding RIPA buffer and boiling for 5 min. Antibodies forimmunoprecipitation were as follows: anti-Ago1 monoclonal Ab (clone 1F2;Wako), anti-Ago2 monoclonal Ab (clone 2D4; Wako), and anti-Ago3monoclonal Ab (clone 1C12; Wako).

Example 1 GAPmer Antisense Oligonucleotides Inhibit leuCAG3tsRNA

GAPmer oligonucleotides were designed to comprise a series of 5′ and 3′locked nucleic acids (LNAs) flanking a sequence of DNA nucleotides(Table 5). GAPmer oligonucleotides provided in Table 5 are complementaryto the tRNA structure shown in FIGS. 2A and 2B.

TABLE 5 GAPmer oligonucleotides. Nucleotides with capitalletters are LNA nucleotides and nucleotides withlowercase letters are DNA nucleotides. SEQ ID NO name complementary tosequence  9 AspPM AspGTC tRNA CCCgtcggggaatcgaACC 10 GL2b GL2b (fire flyTCGaagtattccgcgtACG luciferase) 11 leu3ts leuCAG3tsRNAGGTGtcaggagtggGATT 12 leu5ts leuCAG5tsRNA GACCgctcggccatccTGAC 13 leuAleuCAGtsRNA ACGCagcgccttagACCG 14 leuB leuCAGtsRNA GACTgcgacctgaacGCAG15 leuC leuCAGtsRNA CGCCtccaggggagACTG 16 leuD leuCAGtsRNAGGGAttcgaacccacGCCT 17 leuD-6bp leuCAGtsRNA CGAAcccacgcctccAGGG 18leuD-4bp leuCAGtsRNA TTCGaacccacgcctCCAG 19 leuD-2bp leuCAGtsRNAGATTcgaacccacgcCTCC

Different GAPmer oligonucleotides were tested for the ability to inhibitleuCAGtsRNA. Total RNA was purified by Trizol at 48 hours aftertransfection into HeLa or 293 T cells using 10 nM of each GAPmeroligonucleotide shown in Table 5. 5 μg of total RNA was loaded on 15%denaturing gels and transferred to positively charged nylon membranes.The results demonstrate that the leu3ts GAPmer oligonucleotideeffectively inhibits leuCAG3tsRNA (FIG. 2). The leu3ts GAPmeroligonucleotide bindings to leu3tsRNA and cleaves the target by RNAse Hmediated degradation.

Example 2 MIXmer Antisense Oligonucleotides Inhibit leuCAG3tsRNA

Mixmer oligonucleotides were designed to comprise LNAs interdigitizedwith DNA nucleotides (Table 6). Mixmer oligonucleotides provided inTable 5 are complementary to the tRNA structure shown in FIG. 2A.

TABLE 6 MIXmer oligonucleotides. Nucleotides with capitalletters are LNA nucleotides and nucleotides withlowercase letters are DNA nucleotides. SEQ ID NO name complementary tosequence 1 GL2c GL2 (fire fly GtaCgCgGaaTaCTtC luciferase) 2 leu3ts15PMleuCAG3tsRNA tGTcAGgAgTggGaT (CAGPM) 3 leu3ts15MM1 leuCAG3tsRNAtCTcACgAgTggGaT (CAGMM1) with 2nt mismatch 4 leu3ts15MM2 leu3CAG3tsRNAtGTcAAgAcTggGaT (CAGMM2) with 2nt mismatch 5 leu5ts15PM leuCAG5tsRNAgcTcgGcCaTCcTgA (CAG5ts) 6 CAGcodon leuCAG tRNA GaGacTgcGAccTGacodon region 7 ser15GCTPM SerGCT3tsRNA cgACgAGgAtGggAt (SerPM) 8meti15PM Meti3tsRNA TagCaGAgGAtgGTt (MetiPM)

Different Mixmer oligonucleotides were tested for the ability to inhibitleuCAG3tsRNA. The Mixmer oligonucleotides shown in Table 6 weretransfected at 60 nM into HeLa and HCT cells. Northern hybridizationanalysis was performed 24 hours later. 5 μg of total RNA was loaded on15% denaturing gels and transferred to positively charged nylonmembranes. U6snRNA was used as a loading control. The resultsdemonstrate that the CAGPM oligonucleotide effectively inhibitsleuCAG3tsRNA (FIG. 3). CAGPM binds leu3tsRNA and prevents the target RNAfrom functioning with other molecules (FIG. 3).

Example 3 Mode of Oligonucleotide Action Compared to Known SilencingPathways

The MIXmer oligonucleotides shown to effectively inhibit leuCAG3tsRNAwere tested for activity through known gene silencing pathways. Aluciferase assay was performed at 24 hours after co-transfection ofindicated luciferase construct and 60 nM of oligonucleotide. Eachexperiment was performed in triplicate. The graph shown in FIG. 4 showthat the CAGPM oligonucleotide did not affect gene expression containingperfect complementary sites in its codon or 3′UTR of leuCAG3tsRNA.

LeuCAG3tsRNA was tested to determine if leuCAG3tsRNA has trans-genesilencing activity similar to known microRNA mechanisms. Luciferaseassays were performed at 24 hours after co-transfection of indicatedluciferase construct and 60 nM of oligonucleotide. The results show thatleuCAG3tsRNA does not have trans-gene silencing activity similar toknown microRNA mechanisms (FIG. 5). The graph in FIG. 5 shows thenormalized Renilla luciferase activity. Each construct had two copies ofperfect complementary sites of leuCAG3tsRNA or leuCAG5tsRNA.

LeuCAG3tsRNA was tested to determine whether endogenous genes withsimilar target sequences would be affected. The epoxide hydrolase genehas 1 mis-match complementary site in its open reading frame. Aftertransfection of each 60 nM of indicated oligonucleotide, total extractwas prepared after 24 hours and loaded on SDS PAGE gels. Immunoblot wereperformed with the indicated antibody. The results show thatleuCAG3tsRNA did not affect endogenous genes with similar targetsequences.

Example 4 Inactivation of leuCAGtsRNA Impairs Cell Viability

Cell proliferation was monitored using an MTT assay kit 72 hours aftertransfection of 60 nM of each antisense oligonucleotide in HeLa cellsindicated in FIG. 7A. All experiments were performed in triplicate. TheCAGPM oligonucleotide effectively impaired cell viability in HeLa cells.

Cell proliferation was monitored using an MTT assay kit 72 hours aftertransfection of 60 nM of each antisense oligonucleotide in HCT cellsindicated in FIG. 7B. All experiments were performed in triplicate. TheCAGPM oligonucleotide effectively impaired cell viability in HCT cells.

The CAGPM oligonucleotide specifically impaired cell viability amongseveral different oligonucleotides. Cell proliferation was monitoredusing an MTT assay kit 72 hours after transfection of 60 nM of eachantisense oligonucleotide in HCT cells indicated in FIGS. 7C and D. Allexperiments were performed in triplicate.

To validate the MTT assay results, cell numbers were counted aftertransfection of each oligonucleotide. After transfection of each 60 nMof indicated oligonucleotide, the cell number was counted using ahematocytometer at the day indicated. All experiments were performed intriplicate at results of validation shown in FIG. 8A.

Example 5 Inactivation of leuCAGtsRNA Induces Apoptosis

The effect of antisense oligonucleotides against leuCAGtsRNA onapoptosis was determined. Poly (ADP-ribose) polymerase (PARP) is afamily of protein involved in DNA repair and apoptosis. A key initiationelement of the apoptotic pathway is the activation of caspases followedby cleavage of caspase substrates. Detection of an 89 kDa or 24 kDacaspase cleavage fragment of PARP-1 by caspase 3 and 7 is a hallmark ofapoptosis. The apoptotic markers PARP, Casp-3, 7, and 9 were measuredfollowing transfection of 60 nM of each indicated oligonucleotide.Protein extracts were prepared at each indicated day. The CAGPMoligonucleotide induces apoptosis as indicated by cleaved PARP, Caspase7, and 9 (FIG. 8B).

Annexin V and propidium iodide (PI) was measured to determine apoptoticeffects of different antisense oligonucleotides. Other morphologicalfeatures of apoptosis include loss of plasma membrane asymmetry andattachment, condensation of the cytoplasm and nucleus, andinternucleosomal cleavage of DNA. The membrane phospholipidphosphatidylserine (PS) is translocated from the inner to outer leafletof the plasma membrane. Annexin V is a 35-36 kDa Ca2+ dependentphospholipid-binding protein that binds to cells with exposed PS. Viablecells with intact membranes exclude PI, whereas membranes of dead anddamaged cells are permeable to PI.

Cells were stained with annexin V and PI at each day after transfectionof 60 nM of indicated antisense oligonucleotide. Apoptosis was measuredusing flow cytometry. All experiments were performed in triplicate. FIG.9 show that CAGPM induces apoptosis as determined by annexin V and PIstaining.

Cells were stained with Brdu and PI at 24 hours after transfection of 60nM of each indicated oligonucleotide and cell cycle analysis wasperformed using flow cytometry. The results shown in FIG. 11 show thatthe G1 phase is accumulated in HCT cells after inactivation ofleuCAG3tsRNA by the CAGPM oligonucleotide.

TUNEL assays were performed to measure apoptotic cell death. Cells weregrow in DMEM media with 100% FBS before treatment with 60 nM of theindicated oligonucleotides using lipofectamine transfection. The TUNELassay was performed at 24 hours (FIG. 10A) and 48 hours (FIG. 10B)post-transfection. The results show that CAGPM induces apoptosis.

Example 6 Effect of Antisense Oligonucleotides on Global Translation

The effect of oligonucleotides against leuCAGtsRNA on global translationwas determined. Different numbers of cells were grown in mediumcontaining S35 methionine for 10 minutes at 24 hours after transfectionof each 60 nM of indicated oligonucleotide, and protein extracts wereprepared in RIPA buffer. 10 μg of protein extract for eacholigonucleotide were run on an SDS PAGE gel, stained with coomassieblue, and exposed on a phosphor screen. The results show that globaltranslation is not repressed by inactivation of leuCAG3tsRNA (FIG. 13).

Cells were also treated with cycloheximide to determine the effect ofleuCAGtsRNA downregulation on global protein function. Cells were grownin DMEM with 10% FBS before lipofectamine transfection with indicatedoligonucleotides, and protein synthesis assays were performed 16 hoursafter transfection and 30 minutes after cycloheximide treatment. FIG.12A and FIG. 12B show that downregulation of leuCAG3tsRNA by CAGPM doesnot affect global translation in HeLa and HCT116 cells, respectively.

The effect of codon modification was determined to investigateproduction of proteins having specific codons. Luciferase constructswere designed to detect the effect of anti-leuCAG3tsRNA oligonucleotideson serCAG tRNA function. The leucine codon CTG on the Renilla gene wasreplaced by the alternative leucine codon CTT or CTC to avoid usingleuCAG tRNA. Renilla gene expression was compared in various treatmentconditions by taking the ratio of modified v. original construct. Cellswere grown in DMEM with 10% FBS before transfection with psiCHECK2dual-luciferase constructs. The oligonucleotides were administered 24hours later. Cells were lysed 24 hours after transfection to performdual luciferase assays. Renilla luciferase activity was normalized byFirefly luciferase. The results (FIGS. 14A and 14B) show thatdownregulation of leuCAGtsRNA does not affect the production of proteinscontaining the leucine tRNA.

Example 7 LeuCAG3′tsRNA is an Essential Non-Coding RNA for CellViability

In order to begin to dissect the biological role of 5′tsRNAs and3′tsRNAs (type I tsRNAs) in mammals, the abundance of specific tsRNAspecies was reduced using complementary locked nucleic acid/DNA-mixedantisense oligonucleotides (LNA) (FIG. 3A). The LNA forms a highlystable complex with the target RNA in a sequence specific manner,essentially inhibiting its ability to interact with their biologicaltargets (Jepsen et al., 2004; Kurreck et al., 2002). These molecules arecommonly used for inhibition of microRNA activity in vivo and in vitro(Elmén et al., 2008a; 2008b; Obad et al., 2011). After LNA-mediateddepletion of LeuCAG, SerGCT, AspGTC, and MetCATi (initiator) 3′tsRNA inHeLa and HCT-116 cell lines, reduction of the LeuCAG3′tsRNA (CAGPM) wasobserved, but none of the others nor the GL2 control caused reduced cellviability (FIGS. 3B and 19A), using the MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)cell viability assay (Berridge et al., 2005).

To examine the effect observed with the LeuCAG3′tsRNA-specific LNA(CAGPM), two different 2-nt LeuCAG3′tsRNA mismatched LNAs (CAGMM andCAGMM2) were included in a set of cell viability assays (FIGS. 3C and19B) and northern hybridization (FIG. 3D). The mismatched LNAs, andcontrol (GL2) had no effect on cell viability (FIGS. 3C and 19B) and didnot bind to LeuCAG3′tsRNA, whereas the CAGPM was bound to LeuCAG3′tsRNA,as established by the lack of a northern signal due to the strongassociation of LNA and tsRNA (FIG. 3D).

In concordance with the MTT assay, it was found that a reduction in thenumber of viable cells (determined by cell counting) in both HeLa andHCT-116 cell lines in response to the CAGPM (LeuCAG3′tsRNA knockdown),but not the GL2 and CAGMM LNAs (FIGS. 3F and 19E).

To examine whether the phenotypic effect of the LNAs was the result oftsRNA, but not of mature tRNA sequestration, northern blot analysis wasperformed. While RNA blots can indicate the specificity of LNAs, theycan be misleading because LNAs may block the binding of competing probesas shown in FIG. 3D. The mature LeuCAG tRNA from CAGPM-transfected HeLacells was not detected by the northern probe complementary to theLeuCAG3′tsRNA (FIG. 3D), while a northern probe complementary to the 5′end of LeuCAG tRNA detected two different sized mature tRNAs, the largerband presenting LeuCAG tRNA-LNA complexes (Elmén et al., 2008b; Lanfordet al., 2010). Other mature tRNAs such as SerGCT and MetCATi, weresimilarly detected as two bands after transfection of SerGCTPM andMetCATiPM respectively (Fig S1C). Since no change in cell viability wasobserved when LNAs complementary to the 3′tsRNAs derived from SerGCT,AspGTC, or MetCATi tRNAs, (SerPM, AspPM, and MetiPM)-were transfectedinto cells (FIG. 3B), these results suggest that LNAs directed againsttsRNAs did not functionally affect the general tRNA pool and put intoquestion the interpretation of the northern results suggesting anassociation of the LNA and highly structured mature tRNA in cells (FIG.3D).

Multiple additional approaches were used to further establish that theanti-LeuCAG3′tsRNA LNAs had no effect on the mature tRNA function inliving cells. First, LNA/DNA gapmer antisense (Gap-3tsPM)oligonucleotides were used that, unlike their mixmer counterparts, caninduce RNAse H-mediated cleavage of the target RNA (Kurreck et al.,2002). A gapmer LNAs against the 5′tsRNA (Gap_5′tsPM), anti-codon(GAP_codonPM), and 3′tsRNA (Gap_3′tsPM) region of LeuCAG tRNA wasdesigned (FIG. 3E). It was found that the LNA gapmer directed againstthe 3′tsRNA only eliminated the LeuCAG3′tsRNA and not the correspondingmature tRNA (FIG. 3E), indicating that the LNA is unlikely to beassociated with highly structured mature tRNA inside cells. It was alsofound that when the CAGPM (LNA mixmer) was added into the total RNAextract, it competed with the northern probe interfering with theability to detect a tRNA signal by northern blotting (FIG. 19D). Theseresults confirm that the CAGPM (LNA mixtmer) can inactivate the tsRNAbut not mature tRNA inside cells.

The question of whether LNAs directed against other regions of themature LeuCAG tRNA might interfere with its function was also addressed.Specifically, LNAs complementary to the 5′end and anticodon region ofmature LeuCAG tRNA (CAG5′tsPM and CAGcodon) were transfected into cells(FIGS. 3B and 19B). Neither CAG5′tsPM nor CAGcodon LNAs impaired cellviability (FIGS. 3C). Second, it was possible to reverse the cellviability phenotype resulting from LeuCAG3′tsRNA depletion by additionof an 18nt, 21nt but not 26nt single strand RNA corresponding to the 3′end of the LeuCAG tRNA (FIGS. 3G). The fact that the 26 nt RNA did notreverse the phenotype strongly suggests the phenotypic correction withthe 18 nt and 21 nt RNAs did not result from competitive binding withthe complementary LNAs. Third, there were no LNA-induced changes inglobal protein synthesis determined by using two independent amino acidincorporation assays, Click-iT® AHA chemistry (FIGS. 13A and 20A) and³⁵S-methionine incorporation (FIG. 13B). Fourth, the LeuCUG codons weresubstituted with Leu non-CUG codons in the coding region of thepsicheck2 Renilla gene while leaving the firefly luciferase geneunmodified. Thirteen of the 22 Leucine codons are recognized by theLeuCAG tRNA. After replacement of CUG with non CUG-leucine (e.g. CUC)codons, it was found that Renilla luciferase expression was similarregardless of the LNA (controls vs CAGPM) used for transfection (FIG.13C). All of these experiments showed that the CAGPM LNA does not affectthe function of mature tRNA, and that the inactivation of theLeuCAG3′tsRNA was responsible for the observed decrease in cellviability.

Example 8 Depletion of LeuCAG3′tsRNA Induces Apoptosis

The finding that LeuCAG3′tsRNA depletion caused the cells in culture toround up and detach from the plate (data not shown) suggested anapoptotic process (Falcieri et al., 1994).

To distinguish whether LeuCAG3′tsRNA depletion caused apoptosis ordecreased cell proliferation, three different apoptosis assays wereperformed: Annexin V and PI (Propidium iodide) staining, Tunel (TerminaldUTP Nick End-Labeling), and PARP (poly(ADP-ribose) polymerase) cleavage(reviewed in (Elmore, 2007)) assays. After LNA transfection, cells werestained with Annexin V and PI, and analyzed by flow cytometry. (FIGS.9A, 9B, and 21A). Three days after transfection, the percentage ofLeuCAG3′tsRNA depleted HeLa cells (CAGPM) undergoing early and lateapoptosis were 21.5±4.6% and 24.5±2.7, respectively, compared to5.7±1.4% and 4.6±1.2% from control cells (GL2) (FIGS. 9A and 9B). Anincrease in apoptotic cells was also observed from LeuCAG3′tsRNAdepleted HCT-116 cells (CAGPM) (FIG. 21A). Induction of apoptosis wasfurther confirmed in both HeLa and HCT-116 cells by TUNEL (FIGS. 9B and21B), and PARP cleavage (FIG. 9C) assays. This data strongly suggeststhat the inactivation of this specific tsRNA impairs cell viability byinducing apoptosis.

Example 9 LeuCAG3′tsRNA does not Induce Gene Repression Activity by amiRNA-Mediated Mechanism

A recent report has suggested that tRNA-derived RNAs had a modest effecton mRNA expression through a miRNA-mediated mechanism (Maute et al.,2013). To determine whether the LeuCAG3′tsRNA might function through asimilar process, two different experiments were pursued.

First, a luciferase assay was performed after co-transfection of an LNAand a luciferase vector containing two perfect complementary sequencesof the LeuCAG3′tsRNA in tandem within the 5′UTR or 3′UTR of luciferasegene (FIG. 15A). Regardless of the location of the complementary targetsequence in the mRNA neither the CAGPM nor the GL2 control LNA had aneffect on luciferase expression. Similar results were obtained withother LNAs and their corresponding targets AspGTC and SerGCT3′tsRNApositioned in the 3′ region of the luciferase mRNA (FIG. 22A).Furthermore, co-immunoprecipitate the 5′ and 3′tsRNAs from LeuCAG,SerGCT, and AspGTC tRNA with endogenous Ago 1, 2, and 3 proteins did notoccur suggesting that these tsRNAs are not associated with Ago proteins(data not shown). Collectively, these results suggest that at least asubset of 5′ or 3′ tsRNAs do not have trans-gene silencing activitythrough an Ago-mediated miRNA base-pairing mechanism.

Example 10 Depletion of Leu3tsRNA does not Change Steady State Level ofGlobal Gene Expression

In order to evaluate the effect of tsRNA depletion on global geneexpression, RNAseq on LNA-treated cells was performed (FIG. 15B).High-throughput RNA sequence data was compared from HeLa and HCT-116cells treated with the two control LNAs, GL2 and CAGMM, and the specificLNA against LeuCAG3′tsRNA (CAGPM) (FIG. 15B). Of a total of 30-40million and 18-24 million, 50 base pair paired-end reads from HeLa andHCT-116 cells, respectively, ˜75% mapped to the human genome (Table 7).The mRNA expression patterns were similar between the two control (GL2and CAGMM) LNA-treated HeLa or HCT-116 cells with an r value (Pearsoncorrelation coefficient) of 0.9945 and 0.9916 respectively (FIG. 15B).

TABLE 7 Sequenced sample from FIG. 15. Cell Line Hela HCT-116Transfected LNA GL2 CAGMM CAGPM GL2 CAGMM CAGPM # of Raw reads 3960787633641365 30592282 23563831 22706074 18113883 # of genomic mapped reads30289377 25684785 22952197 17846458 17129415 13408081 % of mapped reads76.5 76.3 75.0 75.7 75.4 74.0

The mRNA expression patterns in CAGPM versus control LNA transfectedHeLa and HCT-116 cells were remarkably similar with only a small numberof differences. The r value between GL2-CAGPM and CAGMM-CAGPM was 0.9882and 0.9868 from HeLa, and 0.9883 and 0.9863 from HCT-116 cells,respectively. Only the PRDM10 gene was consistently up-regulated inCAGPM vs control LNA-transfected HeLa and HCT-116 cells (FIG. 26). Totest whether the CAGPM affected PRDM10 expression, the PRDM10 5′UTR and3′UTR of the PRDM10 mRNA was cloned into the 5′UTR or 3′UTR ofluciferase gene and found, as done in earlier experiments, that theCAGPM LNA had no effect on luciferase activity (FIG. 22B). Furthermore,siRNA-mediated knockdown of the PRDM10 gene did not induce apoptosis(FIG. 17C). Therefore, it appears unlikely that the LeuCAG3′tsRNA has amajor effect on gene expression at the mRNA level.

Example 11 Polysomal Distribution of tsRNAs

Previous studies suggested that tsRNAs are primarily localized in thecytoplasm (Haussecker et al., 2010). Because of the role that tRNAs playin translation, experiments were designed and perfomed to determine ifthe tsRNAs are associated with actively translated mRNAs by examiningtheir distribution following polysome gradient fractionation.Cytoplasmic extracts from cycloheximide-treated HeLa cells wereseparated in 10-50% sucrose gradients, fractionated, and each fractionwas subjected to sequential RNA northern blot analysis. Thesedimentation of a subset of mature tRNAs, microRNAs, GAPDH mRNA(messenger RNA), and tsRNAs was examined (FIG. 15C). GAPDH mRNA, matureLeuCAG and MetCAT e (elongator) tRNA were predominantly associated withthe heavy polysome fractions, while the mature MetCAT i (initiator) tRNAwas predominantly associated with the 80S initiation complex, asexpected. In addition, the sedimentation of microRNAs let-7, miR-15, andmiR-92a showed a similar distribution to the mature tRNAs and GAPDHmRNAs, suggesting that microRNAs are predominantly associated withpolysomal mRNAs. While the 5′tsRNAs from LeuCAG and SerGCT tRNA, and3′tsRNAs from LeuCAG, SerGCT, AspGTC, and MetCATi were predominantlyfound in the light sucrose fractions, the LeuCAG3′tsRNA was distributedbetween both the light and heavy sucrose gradient fractions (FIG. 15C).The functional significance of the observed distribution pattern is notknown.

Example 12 Depletion of LeuCAG3′tsRNA Impairs Ribosome Biogenesis

The cellular polysome profile after depletion of LeuCAG3′tsRNA wasstudied. Remarkably, the proportion of the 40S and 80S ribosomalcomplexes was strikingly decreased, while the relative abundance of the60S subunit was increased in CAGPM versus the GL2 LNA-treated cells(FIG. 16A). To determine the relative abundance of total 40S and 60Sribosomal subunits in the LNA-treated cells, cell lysates were treatedwith puromycin to dissociate 80S and polysomal ribosomes into free 40Sand 60S subunits (Blobel and Sabatini, 1971) (FIG. 16B). InCAGPM-treated cells the amount of 40S subunits was substantiallydecreased to 58.1±1.1%, whereas the amount of 60S subunits were90.7±5.7% of the control (GL2). In agreement with the decrease in 40Sribosomal subunits 18S mature rRNA (ribosomal RNA) abundance wasstrikingly decreased (FIG. 16C).

The decrease in 40S ribosomal subunits only occurred with theanti-3′tsRNA, CAGPM LNA. LNAs directed against the CAG5′tsPM (directedto 5′ end of LeuCAG tRNA), SerPM (directed to 3′ end of SerGCT tRNA), orMetiPM (directed to 3′ end of Meti CAT tRNA) (FIG. 23A) did not alterthe polysome profile.

To distinguish whether the CAGPM LNA reduced rRNA transcription oraffected pre-rRNA processing, the different pre- and mature rRNAsequences by northern blotting using several antisense oligo probes wasexamined (FIG. 16D). Following depletion of LeuCAG3′tsRNA, the 30Spre-rRNA accumulated, while the 41S, 21S and 18S-E pre-rRNA signals weredecreased. In addition, the LNA did not significantly affect 28S maturerRNA processing, and the 45S pre-rRNA signal was unchanged (FIG. 16E).This result was consistent in both HCT-116 and 293 cells (FIG. 23B). Theaccumulation of 30S and the reduction of both 21S and 18S-E pre-rRNAscombined with unchanged steady-state levels of the 45S primarytranscript suggest that removal of the 5′ETS from the 30S intermediateis impaired, hence leading to the reduction in the 40S ribosomalsubunits.

Interestingly, while reduction of the endonuclease and exonucleaserequired for rRNA processing, including XRN2, did not specifically block5′ETS processing (FIG. 16E) (Sloan et al., 2013), the accumulation of30S and the reduction of 21S and 18S-E, and 18S rRNA levels, has beenreported to occur when expression of specific small ribosomal proteins(RPS) such as RPS6, 7, 13, 16, 24, or 28 is reduced (Choesmel et al.,2008; Flygare et al., 2007; Robledo et al., 2008).

To examine a correlation between RPS abundance and LeuCAG3′tsRNAdepletion on ribosomal RNA processing, siRNAs were used to deplete RPS6,RPS10, RPS13, or RPS28. As controls, siRNAs that target the largeribosomal protein RPL7, XRN2, PRDM10, or c-MYC were used (FIG. 16E). TheXRN2, 5′-3′ exonuclease, is required for rRNA processing, while PRDM10was the only gene up-regulated in LeuCAG3′tsRNA depleted HeLa andHCT-116 cell lines. C-MYC was included because it is another regulatorof ribosome biogenesis (Arabi et al., 2005; Grandori et al., 2005;Grewal et al., 2005; Oskarsson and Trumpp, 2005). Depletion of RPS6,RPS13, or RPS28 phenocopied the 5′ETS processing defect (FIG. 16E),while knockdown of RPS10, RPL7, XRN2, PRDM10, and c-MYC did not (FIG.16E). The finding that reducing LeuCAG3′tsRNA and/or a subset of the RPSproteins have similar phenotypic effects suggests the possibility thatthe effects could be interrelated.

Example 13 Depletion of LeuCAG3′tsRNA Down Regulates RPS28 ProteinAbundance at a Post-Transcriptional Step

A computational prediction method using an RNA-hybrid program(Rehmsmeier et al., 2004) using a minimum free energy betweenLeuCAG3′tsRNA and 45S pre-rRNA revealed five, one, one, three and oneputative LeuCAG3′tsRNA binding sites positioned in the 5′ETS, ITS1,ITS2, 28S rRNA, and 3′ETS, respectively (FIG. 24A). Two putative CAGPMLNA binding sites were also found to be nearly complementary to 45Spre-rRNA. To establish if binding to these regions of the rRNAs affectedrRNA processing, LNAs against eleven of the putative tsRNA binding sitesand two of the CAGPM LNA binding sites were designed. None of these LNAsaffected 18S rRNA processing (FIG. 24B) suggesting that neither theLeuCAG3′tsRNA nor the CAPGPM LNA were binding to the rRNA LNA werebinding to the rRNA precursors.

Next, the abundance of several ribosomal proteins were measured afterdepletion of LeuCAG3′tsRNA (FIG. 17A) by western blot. RPS15, 17, 19,25, and 26 were excluded because they are not known to be associatedwith the 5′ETS processing defect. All of RPL proteins were also excludedsince they are required for 28S not 18S rRNA maturation (Choesmel etal., 2008; Doherty et al., 2010; Flygare et al., 2007; Robledo et al.,2008).

Of the ribosomal proteins evaluated only RPS28 was notably downregulated after depletion of LeuCAG3′tsRNA, while RPS10 and RPS16 wereonly slightly reduced. RPS6, RPS7, RPS13, RPS19, RPS24, RPL13a, RPL7,RPL26, and XRN2 were unchanged (FIGS. 17A and 17B). Thenuclear-cytoplasmic localization of RPS6 and RPS28 was also not altered(FIG. 17B). Reduction in the expression of some RPS, including RPS19,RPL11, and RPS13 or other defects in rRNA biogenesis are known to causeapoptosis (reviewed in (Dianzani and Loreni, 2008; Donati et al., 2012;Holmberg Olausson et al., 2012)). It was also found by a PARP cleavageassay that knockdown of RPS10, RPS13, or RPS28 induced apoptosis (FIG.17C). However, RPS10 and RPS13 were eliminated from further studybecause RPS10 is not required for 5′ETS processing (Doherty et al.,2010), and the amount of RPS13 remained unchanged after depletion ofLeuCAG3′tsRNA (FIG. 17A). Since the reduced levels of mature 18S rRNAinduced by depletion of the LeuCAG3′tsRNA was recovered byover-expressing RPS28 (FIG. 17A), the rRNA processing was examined bymeasuring the relative abundance of 18S rRNA precursors (FIGS. 17E and24C). CAGPM caused a 62% and 72% drop in 21S and 18S-E precursors,respectively. Over-expression of RPS28 increased the levels of 21S and18S-E by 26% and 45%, respectively (FIGS. 17E and 24C). This togetherwith the partial recovery of 18S rRNA strongly suggests that it is theloss of RPS28 protein that causes the disruption of ribosome biogenesis.Collectively, depletion of LeuCAG3′tsRNA reduced RPS28 proteinabundance, and disrupted 5′ETS processing during pre-18S rRNAmaturation, either of which can induce apoptosis (FIG. 17C).

Example 14 Depletion of LeuCAG3′tsRNA Suppress RPS28 TranslationalElongation

To determine the cause for reduced RPS28 protein abundance, RPS28 mRNAabundance was measured after transfection of CAGPM LNAs. RPS28 mRNAabundance was not altered (FIG. 17D), even though RPS28 proteinabundance was reduced (FIGS. 17A and 17B). To determine if thetranslation of the RPS28 mRNA was altered by CAGPM, the sedimentation ofmRNAs encoding several ribosomal proteins and another mRNAs (NOP10) wasevaluated with open reading frames of similar length to RPS28 (FIGS.18A-18C). CAGPM transfection selectively shifted the RPS28 mRNA intolighter sucrose gradient fractions compared to the other RPS, NOP10, andGAPDH mRNAs (FIGS. 18A and 18B) suggesting that RPS28 mRNA istranslationally regulated. Based on the polysomal distribution patterntranslation of RPS28 mRNA could be inhibited at the initiation orelongation step. To distinguish between the two possibilities,LNA-treated cells were incubated with harringtonine, a compound thatprevents the first peptide bond formation (Fresno et al., 1977; Ingoliaet al., 2012), thereby essentially freezing the mRNA-ribosome complex inan 80S state at the initiation site (FIG. 25A). If RPS28 mRNAtranslation initiation was suppressed, the RPS28 mRNA should accumulatein sucrose gradient fractions lighter than 80S; however, if elongationwas affected, the RPS28 mRNA would co-sediment with the 80S monosome.The results (FIGS. 18C and 25B) show that upon depletion ofLeuCAG3′tsRNA and harringtonine treatment the RPS28 mRNA accumulates andco-sediments with the 80S monosome (FIG. 18C, fraction 6) stronglysuggesting that RPS28 translation elongation is suppressed.

Results

tRNA-derived small RNAs (tsRNAs) of 18-26 nucleotides in length are anabundant class of small non-coding RNAs. As shown in the examples setforth above, antisense oligonucleotide (LNA)-mediated depletion of aspecific 3′tsRNA, derived from LeuCAG-tRNA, induces apoptosis. Theapoptotic phenotype was reversible by complementation with the specifictsRNA through a non-miRNA-mediated mechanism. Depletion of the tsRNAslowed ribosomal protein S28 (RPS28) mRNA translational elongation andresulted in a decrease in RPS28 protein abundance. Loss of RPS28 proteincaused a block in pre-18S ribosomal RNA processing, decrease in 40Sribosomal subunits, ultimately causing apoptosis. Furthermore, theexamples set forth above show that overexpression of RPS28 inLNA-treated cells restored 18S rRNA processing. These findings indicatethat this particular tsRNA plays an important role in normal ribosomebiogenesis by regulating RPS28 protein abundance, and the onset ofapoptosis. Accordingly, this group of small non-coding RNAs can finetune gene regulation through unique mechanisms.

In the examples set forth above, programmed cell death (PCD) andribosomal biogenesis defect was the result of CAG3′tsRNA depletion.First, the PCD effect was not observed with any of the control LNAs orthose directed against other 5′ and 3′tsRNAs (FIGS. 3B and 3C);secondly, cell viability could be restored with the addition of an 18and 21 bp tsRNA, but not a larger RNA containing the same sequence (FIG.3G); thirdly, LNA treatment had no general effect on protein synthesis,or mature LeuCAG tRNA function in cells (FIGS. 13A and 13B) and on mRNAexpression (FIG. 15B).

Aberrant ribosome biogenesis (nucleolar or ribosomal stress) byinhibition of RNA polymerase I or mutation in ribosome biogenesisfactors, including ribosomal proteins and pre-ribosomal factors cancause cell cycle arrest and apoptosis pathways in a p53-dependent or-independent manner in vivo and in vitro (reviewed in (Deisenroth andZhang, 2010; Dianzani and Loreni, 2008; Donati et al., 2012; HolmbergOlausson et al., 2012)). In addition, knockdown of RPS19 has been shownto impair 18S rRNA maturation and formation of 40S subunit, and toinduce apoptosis in HeLa cells (Choesmel et al., 2007).

The ribosomal protein RPS28 was reduced by the 3′CAGtsRNA or by anindependent method (siRNA) and resulted in a block in 5′ETSpre-ribosomal RNA and induced apoptosis. This together with the findingthat overexpression of RPS28 overcame the block in 18S rRNA productionin LNA-treated cells further supports a cascade of events where3′CAGtsRNA depletion results in a block in RPS28 translationalelongation, reduced RPS28 protein, a block in 5′ETS processing of 45Spre-rRNA, reduced 18S rRNA levels and 40S ribosome subunit production,leading to apoptosis (FIG. 18D).

The importance of RPS28 protein in translation can be inferred from itslocalization to the head of the small ribosomal subunit where itcontacts the 18s RNA and mRNA near or in the exit E-site (Idol et al.,2007; Pisarev et al., 2008). In certain embodiments a ribosome thatlacks RPS28 would have detrimental effects on translation making celldeath a preferred outcome to the production of ribosomes lacking RPS28.As a result, precise regulatory processes have evolved to regulate theproduction of this protein. In fact, other tsRNAs may also have subtleeffects on the expression of other ribosomal proteins that would notnecessarily have a detrimental phenotype in tissue culture cells. Thiswould be consistent with the changing view of the role of ribosome. Itis becoming more clear that ribosomes can no longer be considered aubiquitous and homogenous macromolecular complex with identical functionin all cells. Recent studies suggest developmental and cell typespecific ribosomal proteins may influence the translation of specificmRNAs and promote specific developmental pathways (reviewed in (Xue andBarna, 2012)) sometimes resulting in subtle developmental defects.Aberrant ribosome biogenesis may also play a role in various malignantprocesses (reviewed in (Bywater et al., 2013)). Defects in ribosomebiogenesis are also associated with disease states such as TreacherCollins syndrome (TCS), Shwachman-Bodian-Diamond syndrome (SBDS),Dyskeratosis congenita, 5q⁻ syndrome, and Diamond Blackfan anemia (DBA)(reviewed in (Freed et al., 2010)). Finally, it is also noted that someribosomal proteins may have additional functions outside of their rolewithin the ribosome (reviewed in (Lai and Xu, 2007)).

The data presented herein shows that it is possible to reduce theconcentration of a tsRNA within a cell and induce an apoptotic phenotypewithout interrupting tRNA function, thus providing a new therapeuticapproach for treating human disease.

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The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of thedisclosure, and are not intended to limit the scope of what theinventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the disclosure pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

What is claimed is:
 1. A method of treating cancer in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising an oligonucleotide that targets a specifictRNA-derived small RNA (tsRNA) comprising: (i) 10 to 12 contiguousunmodified nucleic acids having 100% complementarity to the tsRNA and atleast three locked nucleic acids at the 5′ end and the 3′ end of theoligonucleotide, (ii) 15 nucleic acids comprising at least 7 unmodifiednucleic acids and at least 7 locked nucleic acids wherein no more thantwo unmodified nucleic acids are contiguous and no more than two lockednucleic acids are contiguous, (iii) 10 to 12 contiguous unmodifiednucleic acids having 100% complementarity to the tsRNA and at leastthree modified nucleic acids at the 5′ end and the 3′ end of theoligonucleotide, wherein said at least three modified nucleic acids areselected from phosphorothioate nucleic acids, sugar modified nucleicacids, and a combination thereof, or (iv) 15 nucleic acids comprising atleast 7 unmodified nucleic acids and at least 7 modified nucleic acids,wherein no more than two unmodified nucleic acids are contiguous and nomore than two modified nucleic acids are contiguous, and wherein said atleast 7 modified nucleic acids are selected from phosphorothioatenucleic acids, sugar modified nucleic acids, and a combination thereof,wherein the oligonucleotide is complementary to one selected from thegroup consisting of leucine-CAG tsRNA, tsRNA derived from the 5′ end ofmature leucine-CAG tRNA (leuCAG5tsRNA), tsRNA derived from the 3′ end ofmature leucine-CAG tRNA (leuCAG3tsRNA), serine-GCT tsRNA, asparticacid-GTC tsRNA, and methionine-CAT tsRNA, thereby inhibiting viabilityof a cell of the cancer in the subject.
 2. The method of claim 1,wherein the oligonucleotide is selected from the group consisting of:(a) (SEQ ID NO: 11) GGTGtcaggagtggGATT, (b) (SEQ ID NO: 12)GACCgcteggccatccTGAC, (c) (SEQ ID NO: 13) ACGCagcgccttagACCG, (d)(SEQ ID NO: 14) GACTgcgacctgaacGCAG, (e) (SEQ ID NO: 15)CGCCtccaggggagACTG, (f) (SEQ ID NO: 16) GGGAttcgaacccacGCCT, (g)(SEQ ID NO: 17) CGAAcccacgcctccAGGG, (h) (SEQ ID NO: 18)TTCGaacccacgcctCCAG, and (i) (SEQ ID NO: 19) GATTcgaacccacgcCTCC,

wherein uppercase letters represent locked nucleic acids and lowercaseletters represent unmodified nucleic acids.
 3. The method of claim 1,wherein the oligonucleotide is selected from the group consisting of:(a) (SEQ ID NO: 2) tGTcAGgAgTggGaT, (b) (SEQ ID NO: 3) tCTcACgAgTggGaT,(c) (SEQ ID NO: 4) tGTcAAgAcTggGaT, (d) (SEQ ID NO: 5) gcTcgGcCaTCcTgA,(e) (SEQ ID NO: 6) GaGacTgcGAccTGa, (f) (SEQ ID NO: 7) cgACgAGgAtGggAt,and (g) (SEQ ID NO: 8) TagCaGAgGAtgGTt,

wherein uppercase letters represent locked nucleic acids and lowercaseletters represent unmodified nucleic acids.
 4. The method of claim 1,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 5. The method of claim 1, whereininhibiting viability of a cell prevents cell proliferation, inducesapoptosis, or induces cellular necrosis.
 6. The method of claim 5,wherein inhibiting viability of a cell induces apoptosis.
 7. The methodof claim 1, wherein the cell is a human cell.
 8. The method of claim 1,wherein said oligonucleotide is complementary to one selected from thegroup consisting of leucine-CAG tsRNA, leuCAG5tsRNA and leuCAG3tsRNA. 9.The method of claim 8, wherein said oligonucleotide is complementary toleucine-CAG tsRNA.
 10. The method of claim 1, wherein saidoligonucleotide is complementary to leuCAG5tsRNA.
 11. The method ofclaim 1, wherein said oligonucleotide is complementary to leuCAG3tsRNA.12. A method of inhibiting cell proliferation in a subject, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an oligonucleotide that targets a specific tRNA-derived smallRNA (tsRNA) comprising: (i) 10 to 12 contiguous unmodified nucleic acidshaving 100% complementarity to the tsRNA and at least three lockednucleic acids at the 5′ end and the 3′ end of the oligonucleotide, (ii)15 nucleic acids comprising at least 7 unmodified nucleic acids and atleast 7 locked nucleic acids wherein no more than two unmodified nucleicacids are contiguous and no more than two locked nucleic acids arecontiguous, (iii) 10 to 12 contiguous unmodified nucleic acids having100% complementarity to the tsRNA and at least three modified nucleicacids at the 5′ end and the 3′ end of the oligonucleotide, wherein saidat least three modified nucleic acids are selected from phosphorothioatenucleic acids, sugar modified nucleic acids, and a combination thereof,or (iv) 15 nucleic acids comprising at least 7 unmodified nucleic acidsand at least 7 modified nucleic acids, wherein no more than twounmodified nucleic acids are contiguous and no more than two modifiednucleic acids are contiguous, and wherein said at least 7 modifiednucleic acids are selected from phosphorothioate nucleic acids, sugarmodified nucleic acids, and a combination thereof, wherein theoligonucleotide is complementary to one selected from the groupconsisting of leucine-CAG tsRNA, tsRNA derived from the 5′ end of matureleucine-CAG tRNA (leuCAG5tsRNA), tsRNA derived from the 3′ end of matureleucine-CAG tRNA (leuCAG3tsRNA), serine-GCT tsRNA, aspartic acid-GTCtsRNA, and methionine-CAT tsRNA, thereby inhibiting cell proliferationin the subject.
 13. A method of inhibiting viability of a cell in asubject, the method comprising administering to the subject apharmaceutical composition comprising an oligonucleotide that targets aspecific tRNA-derived small RNA (tsRNA) comprising: (i) 10 to 12contiguous unmodified nucleic acids having 100% complementarity to thetsRNA and at least three locked nucleic acids at the 5′ end and the 3′end of the oligonucleotide, (ii) 15 nucleic acids comprising at least 7unmodified nucleic acids and at least 7 locked nucleic acids wherein nomore than two unmodified nucleic acids are contiguous and no more thantwo locked nucleic acids are contiguous, (iii) 10 to 12 contiguousunmodified nucleic acids having 100% complementarity to the tsRNA and atleast three modified nucleic acids at the 5′ end and the 3′ end of theoligonucleotide, wherein said at least three modified nucleic acids areselected from phosphorothioate nucleic acids, sugar modified nucleicacids, and a combination thereof, or (iv) 15 nucleic acids comprising atleast 7 unmodified nucleic acids and at least 7 modified nucleic acids,wherein no more than two unmodified nucleic acids are contiguous and nomore than two modified nucleic acids are contiguous, and wherein said atleast 7 modified nucleic acids are selected from phosphorothioatenucleic acids, sugar modified nucleic acids, and a combination thereof,wherein the oligonucleotide is complementary to one selected from thegroup consisting of leucine-CAG tsRNA, tsRNA derived from the 5′ end ofmature leucine-CAG tRNA (leuCAG5tsRNA), tsRNA derived from the 3′ end ofmature leucine-CAG tRNA (leuCAG3tsRNA), serine-GCT tsRNA, asparticacid-GTC tsRNA, and methionine-CAT tsRNA, thereby inhibiting viabilityof a cell in the subject.